nonenzymatic glycation of type i collagen

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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 267, No. 34, Issue of December 5, pp. 24207-24216,1992 Printed in U.S.A. Nonenzymatic Glycation of Type I Collagen THE EFFECTS OF AGING ON PREFERENTIAL GLYCATION SITES* (Received for publication, June 30, 1992) Karen M. Reiser, MaryAnn Amigable, and Jerold A. Last From the Deoartment of Internal Medicine. School of Medicine, and California Regional Primate Research Center, University of California, Dauis, Califomia 95616-8542A The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr pep- tides, one from each type of chain in the type I tropo- collagen molecule, were investigated in detail: al(I)CB3and a2CB3-5. Togetherthese peptides com- prise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtra- tion, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of a2CB3-5 and al(I)CB3 by bo- ronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and a2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in a2CB3-5,3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly con- served with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites. Aging is characterized by structural changes in the extra- cellular matrix in virtually every tissue and organ system. However, despite a large literature describing the morpholog- ical, physiological, mechanical, and biochemical changes that occur in aging connective tissue, the underlying mechanisms responsible for these changes are still poorly understood. Although for almost 50 years changes in cross-linking have been invoked as playing a key role in connective tissue aging, the specific nature of both the cross-links themselves and the mechanisms responsible for the changes they undergo during aging have only been partially elucidated (1-4). Present knowledge suggests that there are at least two major cross- * This work was supported by a Feasibility Grant from the Amer- ican Diabetes Association (to K. R.) and by National Institutes of Health Grants RR00169 (California Regional Primate Research Ctr. Base Grant), AGO5324 (to K. R.), AGO7711 (to K. R.), and HL32690 (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. linking pathways. In the first pathway, cross-link formation is initiated by the enzyme lysyl oxidase and appears to be tightly regulated, as is apparent in the high degree of tissue specificity of cross-linking patterns and the restriction of cross-linking sites to only a fewwell characterized loci on specific collagen chains (4). The second cross-linking pathway is initiated by the nonenzymatic additionof sugar molecules to lysine and hydroxylysine residues to form Schiff base type adducts. In contrast to enzyme-mediated cross-linking, this stochastic process has no known regulatory mechanisms. Sugar adducts maydirectly affect severalphysicochemical properties of collagen, including conformation, ligand binding, lysyl oxidase-mediated cross-linking, and interactions with other macromolecules, all of whichundergoage-associated changes (1, 5). The sugar adducts are the precursors of the so-called advanced glycation products, or advanced Maillard products. There is a large literature reporting the accumula- tion of these compounds in connective tissue during aging in virtually all tissues and organ systems, as well as their pro- found effect on many physicochemical properties of collagen (1). Ithas also been suggested thattheaccumulation of advanced glycation products may be related to the morpho- logical changes observed in collagen during aging, particularly the loss of structural order in the fibrillar array (1, 6, 7). Despite recent advances in our understanding of nonenzy- matic glycation of proteins, there is relatively little structural information concerning the effects of aging on nonenzymatic glycation of collagen (8). The present study was designed to investigate one aspect of this complex question: the effect of aging on the sites of glucose adduct formation. Our firstgoal was to test the hypothesis that the marked site specificity of nonenzymatic glycation we had observedin collagen from young animals (9) would changewith age, most likely by becoming morerandom as the structural complexity of matrix collagen, which we hypothesized was necessary to direct glu- cose moieties to specific residues, declined. Our second goal was to investigate the relative importance of possible mecha- nisms influencing sites of adduct formation on collagen, in- cluding primary structure, higher order structure, and am- bient glucose concentration. MATERIALS AND METHODS Tissue Source-Tail tendon fibers were obtained from male Spra- gue-Dawley rats aged 6, 18, and 36 months. Tissue was stored frozen at -70 “C until analysis. The sequence of procedures used for prepa- ration of tissue for analysis, described in detail below, is summarized in Fig. 1. Preparation of Purified, “H-Labeled CNBr Peptides-All samples were reduced withNaB“H4 (185 Ci/mol, Amersham)as described previously (10). The effective reducing capacity of NaB3H4 was de- termined using d-amino levulinic acid as a standard compound for reduction (ll), thus allowing ustoconvertthedpm of tritiated glucitolyllysine to molar quantities of glucitolyllysine. The reduced 24207

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Page 1: Nonenzymatic Glycation of Type I Collagen

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 34, Issue of December 5, pp. 24207-24216,1992 Printed in U.S.A.

Nonenzymatic Glycation of Type I Collagen THE EFFECTS OF AGING ON PREFERENTIAL GLYCATION SITES*

(Received for publication, June 30, 1992)

Karen M. Reiser, MaryAnn Amigable, and Jerold A. Last From the Deoartment of Internal Medicine. School of Medicine, and California Regional Primate Research Center, University of California, Dauis, Califomia 95616-8542A ’

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr pep- tides, one from each type of chain in the type I tropo- collagen molecule, were investigated in detail: al(I)CB3 and a2CB3-5. Together these peptides com- prise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtra- tion, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of a2CB3-5 and al(I)CB3 by bo- ronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and a2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in a2CB3-5,3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly con- served with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.

Aging is characterized by structural changes in the extra- cellular matrix in virtually every tissue and organ system. However, despite a large literature describing the morpholog- ical, physiological, mechanical, and biochemical changes that occur in aging connective tissue, the underlying mechanisms responsible for these changes are still poorly understood. Although for almost 50 years changes in cross-linking have been invoked as playing a key role in connective tissue aging, the specific nature of both the cross-links themselves and the mechanisms responsible for the changes they undergo during aging have only been partially elucidated (1-4). Present knowledge suggests that there are at least two major cross-

* This work was supported by a Feasibility Grant from the Amer- ican Diabetes Association (to K. R.) and by National Institutes of Health Grants RR00169 (California Regional Primate Research Ctr. Base Grant), AGO5324 (to K. R.), AGO7711 (to K. R.), and HL32690 (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

linking pathways. In the first pathway, cross-link formation is initiated by the enzyme lysyl oxidase and appears to be tightly regulated, as is apparent in the high degree of tissue specificity of cross-linking patterns and the restriction of cross-linking sites to only a few well characterized loci on specific collagen chains (4). The second cross-linking pathway is initiated by the nonenzymatic addition of sugar molecules to lysine and hydroxylysine residues to form Schiff base type adducts. In contrast to enzyme-mediated cross-linking, this stochastic process has no known regulatory mechanisms. Sugar adducts may directly affect several physicochemical properties of collagen, including conformation, ligand binding, lysyl oxidase-mediated cross-linking, and interactions with other macromolecules, all of which undergo age-associated changes (1, 5 ) . The sugar adducts are the precursors of the so-called advanced glycation products, or advanced Maillard products. There is a large literature reporting the accumula- tion of these compounds in connective tissue during aging in virtually all tissues and organ systems, as well as their pro- found effect on many physicochemical properties of collagen (1). It has also been suggested that the accumulation of advanced glycation products may be related to the morpho- logical changes observed in collagen during aging, particularly the loss of structural order in the fibrillar array (1, 6, 7). Despite recent advances in our understanding of nonenzy- matic glycation of proteins, there is relatively little structural information concerning the effects of aging on nonenzymatic glycation of collagen (8). The present study was designed to investigate one aspect of this complex question: the effect of aging on the sites of glucose adduct formation. Our first goal was to test the hypothesis that the marked site specificity of nonenzymatic glycation we had observed in collagen from young animals (9) would change with age, most likely by becoming more random as the structural complexity of matrix collagen, which we hypothesized was necessary to direct glu- cose moieties to specific residues, declined. Our second goal was to investigate the relative importance of possible mecha- nisms influencing sites of adduct formation on collagen, in- cluding primary structure, higher order structure, and am- bient glucose concentration.

MATERIALS AND METHODS

Tissue Source-Tail tendon fibers were obtained from male Spra- gue-Dawley rats aged 6, 18, and 36 months. Tissue was stored frozen at -70 “C until analysis. The sequence of procedures used for prepa- ration of tissue for analysis, described in detail below, is summarized in Fig. 1.

Preparation of Purified, “H-Labeled CNBr Peptides-All samples were reduced with NaB“H4 (185 Ci/mol, Amersham) as described previously (10). The effective reducing capacity of NaB3H4 was de- termined using d-amino levulinic acid as a standard compound for reduction (ll), thus allowing us to convert the dpm of tritiated glucitolyllysine to molar quantities of glucitolyllysine. The reduced

24207

Page 2: Nonenzymatic Glycation of Type I Collagen

24208 Sites of Nonenzymatic Glycation of Collagen in Aging

I I I

FIG. 1. Preparation and analysis of samples. Scheme showing the collagen sources from which CNBr peptides and tryptic peptides were purified for analysis of extent of glycation and glycation sites ( A ) ; the sequence of steps used to prepare purified, reduced CNBr peptides ( B ) ; and the sequence of steps (C) used for determining the extent of glycation of all of the major CNBr peptides, the identity of the CB3 tryptic peptides purified by HPLC (both glycated and nonglycated tryptic peptides), and the identity of glycated a2CB3-5 tryptic peptides prepared by Affi-Gel affinity chromatography and HPLC. Italic type indicates material that was glycated in vitro before analysis.

material was digested for 4 h with CNBr in formic acid (12). The CNBr digest was initially chromatographed on a carboxymethylcel- lulose ion exchange column (1.5 X 30 cm) using a NaCl gradient in sodium citrate buffer as previously described (12, 13). Fractions from the peaks containing CB3, CB7, CB8, and a2CB3-5 were pooled as previously described (14), lyophilized, and chromatographed on a Bio- Gel A-1.5m column (Bio-Rad) using Tris-HCl/l M CaC12, pH 7.4, as eluant a t a flow rate of 15 ml/h. Absorbance of the effluent stream was monitored spectrophotometrically a t 230 nm. Absorbance peaks known to contain CB3, CB7, CB8, and a2CB3-5 (14) were pooled and desalted on a Bio-Gel P-6 column (Bio-Rad) using 0.2 M NH4HCO:,/0.5% n-propanol as eluant. These peptides were further purified by chromatography on a reversed-phase column (Vydac 218TP54; Separations Group, Hesperia, CA) using a linear acetoni- trile gradient (0-40% CH:,CN over 90 min) containing 0.01 M hepta- fluorobutyric acid as an ion-pairing agent. Peaks were detected spec- trophotometrically a t 230 nm. Relative elution positions of the pep- tides are shown in Fig. 2. Collagen content of purified CNBr peptides was quantified colorimetrically (15) as 4-hydroxyproline after hy- drolysis for 18 h in 6 N HC1 at 110 "C.

Gel Electrophoresis-Purity of isolated CNBr peptides was assessed by electrophoresis on polyacrylamide gels as described previously (12). Standards prepared from calf skin Type I collagen (Sigma) were used as markers. The stained gels were scanned with a densitometer (12) , and the purity of isolated chains and CNBr peptides was expressed as the percent of stainable material comigrating with the authentic marker peptide.

In Vitro Glycation-Intact tendon fibers and purified al(I)CB3 were incubated with glucose in vitro. Intact tendon fibers were washed overnight at 10 "C in 5 mM sodium phosphate buffer containing 0.9% NaC1, pH 7.4, and then incubated with glucose (500 mg/ml) in 20 mM sodium phosphate buffer containing 3 mM sodium azide at 37 "C for 24 h. The CNBr peptide al(I)CB3 was purified from rat tail t.endon tendon collagen as described above and then incubated with

30 40 5 0 60 7 0

TIME (MINUTES)

FIG. 2. Purification of CNBr peptides by HPLC. Absorbance tracing of CNBr peptides from type I collagen separated by reversed- phase HPLC using a linear acetonitrile gradient (0-40% CH,CN over 90 min) containing 0.01 M heptafluorobutyric acid as an ion-pairing agent. Peaks were detected spectrophotometrically at 230 nm. Frac- tions were pooled as indicated by horizontal bars. The identities of peaks containing CNBr peptides identified by gel electrophoresis (see Fig. 3) are indicated.

glucose under the same conditions used for the intact collagen fibers. Preparation of Tryptic Peptides from CB3"Purified CB3 and

a2CB3-5 were digested with trypsin (~-l-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin immobilized on agarose beads, Pierce) according to the method of Yamauchi et al. (16). Briefly, the samples were dissolved in 0.2 M NH,HC03, pH 7.9, and heated to 65 "C for 20 min with constant stirring. The samples were allowed to cool to 37 "C, and trypsin was added (1% w/w). After 4 h the solution was heated to 60 "C for 10 min. After the samples were again cooled to 37 "C, additional trypsin was added (0.5% w/w) and digestion was allowed to proceed for an additional 2 h.

The tryptic peptides were separated by HPLC' using the method of van der Rest and Fietzek (17). Briefly, samples containing up to 50 pg of hydroxyproline were loaded on a reversed-phase column (Vydac 218TP54; Separations Group), and the peptides were eluted with a linear acetonitrile gradient (4-32% over 90 min) containing 0.01 M heptafluorobutyric acid as an ion-pairing agent. Absorbance of the column eluate was monitored a t 210 nm. Fractions were collected at 1-min intervals, and their radioactivity was measured by removing an aliquot (usually 100 pl) of each fraction for liquid scintillation counting. Peak fractions were pooled and lyophilized.

Hydroxyproline content of the purified tryptic peptides was deter- mined by the method of Einarsson (18). Briefly, aliquots of the purified peptides were hydrolyzed in 6 N HCl a t 110 "C for 18 h, dissolved in distilled water, and brought to a volume of 900 pl in a 5- ml reaction vial (Reactivial; Pierce). Then, 100 pl of 0.8 M borate buffer, pH 9.5, was added, followed by 2 ml of CH&N containing 100 mg of o-phthalaldehyde and 52 p1 of 0-mercaptoethanol to remove primary amines. After 30 s, 2 ml of CH&N containing 140 mg of iodoacetamide was added; after another 30 s 300 pl of 9-fluorenyl- methylchloroformate (5 mmol/ml acetone; Pierce) was added. The resultant solution was extracted twice with diethyl ether. Aliquots of the residue were analyzed by reversed-phase HPLC (Micro-Sorb Short One; Rainin, Emeryville, CA); the mobile phase (flow rate = 2 ml/min) consisted of 35% acetonitrile in water containing 1.2% acetic acid. Derivatized hydroxyproline and proline were detected fluoro- metrically (excitation = 260, emission = 320) on a Hitachi fluorometer (Model 2000; EM Science, Gibbstown, NJ). A standard curve was prepared by derivatizing standards containing known amounts of proline and hydroxyproline (Sigma).

Preparation of Radiolabeled Glucitolyllysine and Glucitolylhydroxy- lysine Standards-Preparation of I4C-labeled standards for glucito- lyllysine and glucitolylhydroxylysine (and their hydrolysis re- arrangement products) were prepared according to the method of Robins (19) and of Yue et al. (20), as previously described (21). Briefly, equimolar amounts of glucose and radiolabeled lysine (1.1 pCi/mg; Amersham) were incubated in neutral phosphate buffer in the presence of NaCNBHl for 24 h at room temperature. At the end of the incubation period HC1 was added to decrease the pH to 2.0 and glucitolyllysine was separated from unreacted lysine by chroma- tography on a boronate affinity column (20). A 1 X 14-cm glass column was packed with a slurry of Affi-Gel 601 (Bio-Rad) in 0.025

' The abbreviations used are: HPLC, high-performance liquid chro- matography; HLNL, hydroxylysinonorleucine.

Page 3: Nonenzymatic Glycation of Type I Collagen

Sites of Nonenzymatic Glycation of Collagen in Aging 24209

M sodium phosphate buffer, pH 9, and equilibrated with the same buffer. After loading the [’4C]glucose-lysine reaction mixture on the horonate column, we washed the column with phosphate buffer followed by 50 ml of 0.025 M HC1. Radioactivity present in the HCI eluate, the fraction containing glycated compounds, was analyzed on a reversed-phase column (Ultrasphere; Altex, Berkeley, CA) under isocratic conditions (mobile phase = 0.01 M sodium phosphate, 0.3% sodium dodecyl sulfate/22% n-propanol, pH 2.84, flow rate = 1 ml/ min). There were two major radioactive peaks, corresponding to lysine glycated at the 01 and 6 sites, respectively. A small peak coeluting with authentic lysine was also present and represented less than 1% of the total unreacted lysine. The bulk of the unreacted lysine did not bind to the column and was present in the phosphate wash. As described by us previously (21), the e-glycated compound was identified by incubating poly(L-lysine) (Pierce) with [‘4C]glucose in order to gen- erate predominantly e-glycated lysine. This preparation had to be hydrolyzed before chromatography, a procedure that generates glu- citolyllysine rearrangement products. Again, the hydrolysate was chromatographed on a boronate column as described above, and the HC1 was eluate analyzed by HPLC. This time the radioactive peak eluted as a doublet, representing glucitolyllysine and its hydrolysis rearrangement product. The unreacted lysine was detected by post- column derivatization with o-phthalaldehyde (21). To confirm the identity of the radioactive peak as e-glycated [14C]glucitolyllysine, an aliquot was subjected to a Smith degradation as described by Robins (19). More than 80% of the counts coeluted with authentic lysine, indicating that glucitolyllysine had been degraded to yield lysine. When an aliquot of purified glucitolyllysine that had not been sub- jected to the Smith degradation was rechromatographed, all of the radioactivity eluted at the glucitolyllysine position, indicating that spontaneous degradation did not occur. Markers for glucitolylhydrox- ylysine were prepared in the same way by incubating [14C]glucose and hydroxylysine for 24 h in the presence of NaCNBH3.

Preparation of Glycated Tryptic Peptides-Glycated tryptic pep- tides were separated from nonglycated peptides by affinity chroma- tography with Affi-Gel 601 using conditions described above. Frac- tions were collected a t 1-min intervals for liquid scintillation count- ing. To confirm that the radioactivity in the HPLC-purified tryptic peptides present in the Affi-Gel retentate was derived only from reductively labeled glucitolyllysine residues, aliquots of each peak were hydrolyzed and analyzed for glucitolyllysine content by HPLC. In all of the samples, the bulk of the radioactivity coeluted with I4C- labeled glucitolyllysine markers. Attempts were also made to separate glycated CNBr peptides from nonglycated peptides using affinity chromatography with Affi-Gel 601; however, these attempts were unsuccessful.

Amino Acid Analysis-Compositional analyses of purified tryptic peptides were performed using a modification of the Waters “Picotag” method (22). This method was chosen for its rapidity (less than 12 min per run) and the fact that phenylisothiocyanate derivatives of secondary amines such as hydroxyproline and proline not only are detectable at the same wavelength as primary amines but also have color yields comparable to those of primary amines. Aliquots of purified tryptic peptides containing 1-2 pg of hydroxyproline (deter- mined as described above) were lyophilized in 6 X 50-mm sample tubes (Corning Glass Works) that had been acid-washed in 6 N HC1. Up to five sample tubes were placed in a 20-ml vacuum hydrolysis tube (Pierce), and 200 pl of constant boiling 5.7 N HC1 (Pierce) containing 1% phenol was placed in the bottom of the hydrolysis tube. This procedure permits vapor phase hydrolysis. The vacuum tube was evacuated and purged with N, for three cycles before being sealed with a Teflon plug and placed in a 106 “C oven for 18 h. After hydrolysis the sample tubes were gently wiped on the outside to remove any condensed HC1 and dried under vacuum. Next, 10 p1 of drying solution (ethanol:triethylamine:water, 2:2:1) was placed in each tube, and the samples were once again dried under vacuum. The samples were derivatized with phenylisothiocyanate (Pierce) by add- ing 20 pl of derivatization solution (ethano1:triethylamine: water:phenylisothiocyanate, 7010:10:10) to each sample tube, vortex- ing gently, and allowing the tubes to stand at room temperature for 20 min. Reagents were then removed under vacuum. Dried samples were stored frozen until chromatography; they are stable for at least 3 weeks. Immediately before chromatography, samples were dissolved in approximately 100 p1 of sample buffer. Reconstituted samples were stable a t 5 “C for approximately 72 h. Aliquots of samples containing 200-400 pmol of hydroxyproline were analyzed on a 1.5 X 10-cm Waters Picotag analysis column heated to 43 “C in a Fiatron column heater (Fiatron Lab Systems, Oconomowoc, WI). Approximately 1 m

of tubing between the injector and the column was looped around the precolumn heating coils in the heater in order to prewarm the mobile phase. The mobile phase consisted of buffer A (sodium acetate trihydrate in 6% acetonitrile containing 0.05% triethylamine, pH 6.38) and buffer B (60% acetonitrile in water). Samples were eluted using a complex gradient recommended for “Picotag” analysis on the Waters Picotag column (22)as follows. 0-10 min: percentage of B increases from 0 to 54% in a convex gradient; 10-11 min: percentage of B increases to 100% in a linear gradient. Derivatized amino acids were detected a t 240 nm on a Waters UV detector Model 440. Peak areas were determined on a Hitachi Model D2000 integrator (EM1 Science). Standards consisted of a commercial mixture of 19 amino acids, including hydroxyproline and hydroxylysine (Pierce).

Sequence Analysis-Tryptic peptides purified by HPLC were se- quenced for four to nine cycles by the University of California at Davis Protein St,ructure laboratory by automated Edman degradation using a gas phase automatic sequenator.

Analysis of Protein Structure-Based on the known primary struc- ture of CB3, selected properties of the peptide were analyzed, includ- ing hydrophilicity, amphiphilicity, and flexibility, using a computer program (MacVector; IBI, New Haven, CT). Hydrophilicity indices were calculated using the Kyte-Doolittle scale (23) with a window size of 8. The hydrophobicity values of the eight amino acids were summed and divided by N to obtain the average hydrophobicity per residue for the window. Final values are expressed as hydrophilicity rather than hydrophobicity, as originally described by Kyte and Doolittle (23).

Statistical Analyses-All analyses were performed on a microcom- puter (Macintosh IIci) using statistical software package (Statview 500; Abacus, Berkeley, CA). Comparisons between three or more groups were made by ANOVA; a significance level of 95% was used to determine significance between groups.

RESULTS

Extent of Glycation of CNBr Peptides-The CNBr peptides al(I)CB3, al(I)CB7, al(I)CB8, and a2CB3-5 were purified from rat tail tendon by the chromatographic procedures de- scribed under “Materials and Methods” (Fig. 1B); the purity of all of the peptides analyzed in this study was >95% as assessed by gel electrophoresis (Fig. 3). We have previously shown that peptides purified by these techniques have com- positional analyses consistent with literature values (14).

To determine the glucitolyllysine content of the purified CNBr peptides, hydrolyzed aliquots were analyzed for gluci- tolyllysine and -hydroxylysine content by HPLC using the conditions described above for analysis of the 14C-labeled markers. As described in detail previously, this technique clearly separates the glucitolyllysines and their hydrolysis rearrangement products from reductively labeled lysyl oxi- dase-mediated cross-links, such as dihydroxlysinonorleucine and HLNL (21). Glucitolyllysine content of each of the puri- fied CNBr peptides, prepared from 6-month-old animals, is shown in Table I. All of the peptides analyzed were glycated. The most glycated of the CNBr peptides analyzed was a2CB3- 5, whether expressed as moles of glucose per mol of peptide, or as percent of lysine residues glycated per peptide.

We selected two peptides for detailed investigation of sites of glucose adduct formation, CB3 from the al(1) chain and a2CB3-5 from the 02 chain. We selected a2CB3-5 in part because it contains a known locus for lysyl oxidase mediated cross-linking; we were interested in determining if the prox- imity of lysyl oxidase cross-link sites might influence nonen- zymatic glycation. Although al(I)CB6 also contains a major locus for lysyl oxidase-mediated cross-linking, it is extremely difficult to purify monomeric CB6 from tissue from older animals because of its participation in high molecular weight cross-linked complexes (24). In contrast, a2CB3-5 is readily purified, even from tissue from older animals. We selected al(I)CB3 as a representative peptide from the al(1) chain because of its ease of purification and its lack of any known or suspected enzymatically mediated cross-linking sites.

Page 4: Nonenzymatic Glycation of Type I Collagen

Sites of Nonenzymatic Glycation of Collagen in Aging

I 1 l , , , 1 1 1 1 1 1 I

20 4 0 6 0 8 0 100 120

DISTANCE FROM TOP OF GEL (mm)

FIG. 3. Analysis of purified CNBr peptides by gel electro- phoresis. Densitometer tracings of CNBr peptides electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels and scanned at 520 nm. A-D, CNBr peptides purified as described under “Materials and Methods”; E, standard, consisting of calf skin type I collagen that was digested with CNBr.

TABLE I Glucitolyllysine content of CNBr peptides purified from rat tail

tendon collagen Data are expressed as moles of glucitolyllysine per mol of peptide.

Values represent the average of two analyses, performed on tissue obtained from two different animals; individual values are shown in uarentheses.

Peptide Glucitolyllysine content Lys residues Percent Of glycated lysines“ mollpeptide %

nl(I)CB3 0.028 (0.034, 0.024) 5 0.56 tul(I)CB7 0.048 (0.040, 0.056) 10 0.48 nl(I)CB8 0.030 (0.029, 0.031) 10 0.30 n2CB3-5 0.127 (0.132, 0.122) 18 0.71 “ Percent of glycated lysine residues = moles of glucitolyllysine/

mol of lysine per peptide) X 100. Average values for the two deter- minations are shown.

Effects of Aging on Extent of Glycation-We measured glu- citolyllysine content of purified CB3, purified a2CB3-5, and unfractionated tail tendon collagen obtained from 6-, 18-, and 36-month-old animals. Samples were prepared from two dif- ferent animals at each time point. We found that there were no significant differences in extent of glycation between young and old animals in either the unfractionated collagen or in the purified peptides (data not shown).

Identification of Tryptic Peptides from CB3 and Analysis of Glycation Sites-Tryptic digests of HPLC-purified CB3 were chromatographed on a C18 reversed-phase column, using the gradient system described by van der Rest and Fietzek (17). Absorbance profiles were monitored at 230 and 1-ml fractions were collected at 1-min intervals for liquid scintillation count- ing. A typical absorbance profile is shown in Fig. 4. Fractions were pooled as indicated and lyophilized. Aliquots of the pooled fractions were hydrolyzed and analyzed for amino acid

m u ) 50 60 70

TIME (Minutes)

FIG. 4. Purification by HPLC of tryptic peptides from al(I)CB3. Absorbance tracing of CB3 tryptic peptides separated by reversed-phase HPLC using an acetonitrile gradient containing hep- tafluorobutyric acid as an ion-pairing agent. Peaks (pooled as indi- cated) were identified by compositional analysis and sequence analy- sis (Tables 11-111).

composition; the remainder of the pooled peaks was used for determining amino acid sequence. We were unable to obtain enough material for sequencing from Peaks I-II. Based on compositional data (Table 11) and sequence data (Table 111), we were able to identify the peptides comprising all of the absorbance peaks except Peak I, and to determine the elution position of all of the lysine-containing tryptic peptides. Peaks IV, VI, and VII contained mixtures of two separate tryptic peptides, as could be clearly seen in the sequence data. In contrast, Peaks VIII and IX, each of which contained both a Lys and an Arg residue, did not contain a mixture of two coeluting peptides, but rather contained a single peptide in which cleavage had not occurred at a lysine residue. Even repeated digestion with trypsin of these peptides yielded no further cleavage at the lysine site. Our results are very similar to those reported by van der Rest and Fietzek (17), including the observation of tryptic peptides containing uncleaved Lys and Arg residues. In addition, our observation that the small absorbance peaks immediately preceding Peak IX had com- positions identical to that of Peak I X (data not shown) was also reported by van der Rest and Fietzek (17).

Once we had identified all of the tryptic peptide peaks, we analyzed the distribution of glucose adducts among the lysine residues. The CB3 tryptic digest was chromatographed on an Affi-Gel affinity column; the retentate (i.e. the glycated tryp- tic peptides) was then chromatographed on a reversed-phase HPLC, and fractions were collected for liquid scintillation counting. A typical radiochromatograph of glycated tryptic peptides prepared from CB3 is shown in Fig. 5. To ensure that all of the radioactivity was derived from reduced sugar adducts, aliquots from each peak were hydrolyzed and ana- lyzed for the presence of glucitolyllysine by HPLC as de- scribed under “Materials and Methods.” In each peak, all of the radioactivity was accounted for by reduced sugar adducts. In addition, we confirmed the identity of the glycated peptides comprising Peaks A, D, and E by compositional analysis (data not shown); these data were consistent with the identification made by sequence analysis of unfractionated material. The peaks in Fig. 5 are labeled with letters to avoid the implication that their constituent peptides are the same as those in the absorbance peaks shown in Fig. 4. The peaks shown in Fig. 5 are comprised only of peptides containing a glycated lysine residue, whereas some of the peaks in Fig. 4 include peptides with no lysine residues.

Of the 5 lysine residues present in CB3, Lys-434 was the preferred site of glucose adduct formation. Lys-434 was pres- ent in two peptides: the small peptide terminating in a Lys

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Sites of Nonenzymatic Glycation of Collagen in Aging 24211

TABLE I1 Amino acid composition of oll(Z)CB-3 tryptic peptides purified by HPLC

Peaks identified by Roman numerals represent pooled fractions as shown in Fig. 4, with the amino acid residues shown in parentheses. Identification of the tryptic peptides was based on both sequence analysis (Table 111) as well as composition. In those peaks in which compositional analysis revealed both a Lys and an Arg residue, sequence analysis permitted the distinction between a peak containing two coeluting tryptic peptides and a peak containing a single tryptic peptide that contained an uncleaved Lys or Arg residue. Literature values for the composition of the presumptive peptides present in each peak are shown in parentheses (39). Based on sequence analysis, the two peptides in Peak VZ were present in equimolar amounts; the peptides present in Peaks I V and V I I were present in approximately a 2:l ratio and a 1:2 ratio. resuectivelv. Values reuresent averages of three determinations.

Peak Residue

I I1 (409-416) I11 (532-551)" Iv (520-531)a V (508-519)" (508-519)a vII (502-507)a VI11 (421-453)" IX (454-498)" (435-453) (421-434) (403-408)

ASP Glu HPr Ser GlY His '4% Thr Ala Pro TY r Val Met Ile Leu HY 1 Phe LY s

2.1 0 1.2 1.3 5.2 0 0.6 0.5 1.8 0.3 0 0 0 0 0 0 0 1

" Identification of the peptides comprising these peaks was confirmed by sequence analysis (Table 111).

TABLE 111 Amino acid sequences of CB3 tryptic peptides separated by HPLC

Peaks identified by Roman numerals represent pooled fractions as shown in Fig. 4. Amino acid residues comprising the peaks are shown in parentheses; compositional analyses of these peaks are shown in Table 11. The number of cycles analyzed for each peak depended on the amount of material available for analysis and the yields. For each peak, however, a sufficient number of residues were analyzed to definitively identify the tryptic peptide(s) present in the peak. In those peaks in which compositional analysis revealed both a Lys and an Arg residue, sequence analysis allowed us to determine if the peak contained two coeluting tryptic peptides or a tryptic peptide that contained an uncleaved Lys or Arg residue. The residues comprising the tryptic peptides in each peak are shown in parentheses.

Cycle Peak

111 (532-551) IV (435-453; 520-531) V (508-519) VI (421-434; 508-519) VI1 (403-408: 502-507) VI11 (421-453) IX (454-498)

1 Gly (900)" Asp + Gly (600 + 400) Gly (500) Gly (5500) Gly (3600) Gly (390) Gly (500) 2 Asp (900) Gly + Asn (500 + 250) Val (425) Val (5500) Phe (3900) Val (330) Glu (425) 3 Thr (800) Glu + Asn (475 + 150) Gln (325) Gln + Hpr (1700 + 1700) Hpr (3000) 4 Gly (800) Ala + Gly (475 + 300) Gly (360) Gly (3700)

Hpr (200) Gln (325) Gly (2550)

5 Ala (800) Gly + Ala (500 + 300) Pro (280) Pro (3500) Gly (270) Gly (360)

Glu + Pro (1600 + 800) Pro (280) 6 Hpr (700) Hpr (2500) " Amino acids were identified by automated Edman degradation; yields for each cycle, expressed as picomoles, are shown in parentheses.

residue (residues 421-434) that comprised Peak B, and the larger peptide (residues 421-453) terminating in an Arg resi- due that comprised Peak D. Of the remaining 4 Lys residues present in CB3, glucose adducts could be detected on Lys-408 (Peak C), Lys-479 (Peak E ) , and Lys-531 (Peak A ) . No glucose adducts could be detected on Lys-416 (the peptide containing Lys-416 elutes at 26 min; it is present in Peak II in Fig. 4).

Identification of Tryptic Peptides f rom a2CB3-5 and Analy- sis of Glycation Sites-We were unable to apply the same strategy used for mapping the CB3 tryptic peptides to analysis of a2CB3-5, a peptide approximately four times larger than CB3. Unfractionated tryptic peptides from a2CB3-5 could not be readily separated by a single HPLC program. However, glycated tryptic peptides from a2CB3-5, prepared by boronate affinity chromatography of the tryptic digest, were readily separated by HPLC. A typical radiochromatograph is shown in Fig. 6. We observed three major radioactive peaks (Peaks

A, B, and D), as well as two relatively minor peaks (C and E ) . Absorbance chromatographs are not shown as there was insufficient material present in these purified, glycated tryptic peptides to permit clear detection of peaks by spectrophotom- etry. As in the case of CB3, we determined that all of the radioactivity present in these peaks was derived from glucose adducts by hydrolyzing aliquots and analyzing them for glu- citolyllysine content. Next, we investigated the fate of the enzyme-mediated cross-link HLNL during the various chro- matographic procedures, as we knew that this reductively labeled compound was present in the purified a2CB3-5 pep- tide. We first determined that only a small percentage (less than 10%) of the total HLNL present on the a2CB3-5 peptide was retained on the Affi-Gel affinity column. We next deter- mined that the elution time of the tryptic peptide containing HLNL was 110 min, considerably later than any peak con- taining reduced glucose adducts (Fig. 6). We determined that recovery of radioactivity from the column was greater than

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24212 Sites of Nonenzymatic Glycation of Collagen in Aging

TIME (Minutes)

FIG. 5. Purification of glycated tryptic peptides from al(I)CB3 by HPLC. Radiochromatograph of glycated tryptic pep- tides prepared by boronate affinity chromatography of a tryptic digest of nl(I)CB3 and then separated by HPLC as described in Fig. 5. Radioactivity in all of the peaks was determined to be attributable only to reduced glucose adducts. Glycated lysine residues present in the peptides comprising each peak are as follows. Peak A , Lys-531; Peak B, Lys-434; Peak C, Lys-408; Peak D, Lys-434; Peak E, Lys- 479.

o . l . . . . . . . , . , . , . , . , . , . , . , I 0 10 2 0 30 4 0 5 0 6 0 7 0 8 0 90 l o 0 110

TIME (Minutes)

FIG. 6. Purification of glycated tryptic peptides from a2CB3-5 by HPLC. Radiochromatograph of glycated tryptic pep- tides prepared by boronate affinity chromatography of a2CB3-5 and then separated by HPLC as described in Fig. 4. Peaks were pooled as indicated and were identified by compositional analysis and sequence analysis (Tables IV-V). Glycated lysine residues present in the pep- tides comprising each peak are as follows: Peak A , Lys-453; Peak B, Lys-479; Peak C, Lys-974; Peak D, Lys-924; Peak E, mixture of several lysine residues, as shown in Tables IV-V.

95%. In addition, we analyzed forepeak radioactivity and washout radioactivity for glucose adducts; no glucitolyllysine was detectable in these fractions. In summary, we determined that the radioactivity in the peaks shown in Fig. 6 was derived exclusively from reduced sugar adducts, that any radioactivity arising from enzymatic cross-links did not coelute with this material, and that the radioactive sample applied to the column was quantitatively recovered. We then pooled the peaks as indicated in Fig. 6 in order to identify their constit- uent peptides. We hydrolyzed aliquots for compositional analysis and used the remainder of the samples for amino acid sequence analysis. These data enabled us to identify the constituent peptides in Peaks A-E (Tables IV and V).

Out of 19 potentially glycatable residues, there were three preferential sites of glycation on a2CB3-5: Lys-453 (Peak A ) , Lys-479 (Peak B ) , and Lys-924 (Peak D). Lys-974 (Peak C) represented a relatively minor glycation site. Peak E com- prised a mixture of glycated tryptic peptides, based on se- quence analysis. The predominant peptide in this peak, based on residue yields obtained during sequence analysis (Table V), suggest that the predominant peptide in Peak E consisted of the same tryptic peptide found in Peak C, residues 964-

974. The remainder of the radioactivity in Peak E was derived from at least three small tryptic peptides: residues 652-654, 655-657, and 652-657. We did not pursue development of HPLC techniques for complete separation of the small tryptic peptides present in Peak E (which would be difficult due to their similar size and composition), as the percentage of the total glucose adducts in a2CB3-5 that was present in Peak E was less than 10%.

Effects of Age on Preferential Glycation Sites-We analyzed the patterns of glucose adduct formation in CB3 and a2CB3- 5 prepared from rats of three different ages: 6, 18, and 36 months. The effects of age on the preferential glycation sites of these type I CNBr peptides are shown in Tables VI and VII. The data are expressed as the percentage of the total glucose adducts present on the peptide that are located at a specific residue. For example, for 6-month-old rats, 71% of all the glucose adducts on CB3 are located at Lys-434 (Table VI). This form of data expression was chosen to more readily permit comparisons among the different age groups. We were surprised to find a remarkable degree of conservation of preferential glycation sites even in extremely old rats. We had initially assumed that nonenzymatic glycation would be af- fected by the changes in higher order structure of collagen known to occur with aging, and that we might observe either alterations in the predominant glycation sites, or possibly increased randomness of sites of glucose adduct formation. To the contrary, there were no changes in the predominant glycation sites with age. However, there was a significant decrease with age in relative glycation of Lys-479, a relatively minor site in CB3 (Table VI). Although there were no statis- tically significant changes in a2CB3-5 with age (most likely because of the small number of determinations), there was a trend toward a decrease in relative glycation of Lys-924, which is the lysine residue closest to HLNL (Table VII).

I n Vitro Glycation-Experiments were then conducted in uitro to investigate mechanisms determining preferential gly- cation sites. We compared glycation sites in the following samples: 1) CB3 isolated from 6-month-old intact tail tendon fibers after the fibers had been incubated with sugar in uitro for 24 h; 2) CB3 purified from 6-month-old tendon fibers followed by incubation of the isolated CB3 with glucose. The steps used for sample purification and preparation are shown in Fig. 1, A and B. We hypothesized that isolated CB3 glycated in uitro would have a more random distribution of glucose adducts than would CB3 prepared from intact tendon fibers glycated in uitro, as there would be no higher order structure to direct sites of glucose attachment during the in uitro incubation. The results, summarized in Table VIII, were surprising. Lys-434 was the preferential locus for glycation in both isolated CB3 glycated in uitro and in CB3 prepared from intact fibers glycated in uitro, just as it had been in the samples that had not been incubated with sugar. Even though isolated CB3 presumably existed as a random coil while it was incu- bated in the sugar solution, glucose still overwhelmingly pre- ferred to form adducts with Lys-434. However, there were significant differences between CB3 that was glycated after it was purified and CB3 that had been glycated while it was still part of the intact tropocollagen molecule. The relative glyca- tion of Lys-434 was significantly increased in the CB3 gly- cated after it was purified (82 us. 71.5% in intact fibrils), and the relative glycation of Lys-531 was significantly decreased (4 us. 13% in intact fibrils).

These experiments with purified CB3 suggested that al- though higher order structure did not play a major role in determining sites of glycation, it appeared to influence the relative distribution of the glucose adducts among these sites

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Sites of Nonenzymatic Glycation of Collagen in Aging 24213

TABLE IV Amino acid composition of (uZ(IiCB3-5 tryptic peptides purified by HPLC

Peaks identified by letters A-E represent pooled fractions shown in Fig. 6; the residues comprising the tryptic peptide present in each peak are shown in parentheses. Identification of tryptic peptides is based on both sequence analysis (Table V) as well as composition. Literature values for each peptides are shown in parentheses. Since rat a2CB3-5 has not been sequenced completely, literature values are those that have been obtained for bovine a2CB3-5 (39).

Amino acid Peak

A (430-453)" B (454-479)" C (964-974)" D (889-924)" E (mixturePb

ASP Glu HPr Ser Gly His Arg Thr Ala Pro TY r Val Met Ile Leu HY 1 P he Lvs

0 0.3 1.1 1 6 0 2 0 2.3 2.4 0

0 1.9

1 0 0 0 1

Identification of the peptides comprising these peaks was confirmed by sequence analysis. *Based on sequence analysis (Table V), peak E contains a mixture of peptides, most likely some or all of the following residues: 652-654,

655-657,652-657,907-924, and 964-974.

TABLE V Amino acid sequences of a2CB3-5 tryptic peptides separated by HPLC

Residues and yields for each cycle are shown for the tryptic peptide peaks shown in Fig. 4; compositional analyses of these peaks are shown in Table IV. The number of cycles analyzed for each peak depended on the amount of material available for analysis and the yields. For each peak, however, a sufficient number of residues was analyzed to definitively identify the tryptic peptide present in the peak. The residue numbers for the tryptic peptides present in each peak are shown in parentheses.

Cycle Peak

A (430-453) B (454-479) C (964-974) D (889-924) E (Mixture)"

Gly (60) Gly (50) Gly (35) Gly (1252) Glu (15) Pro (9) Glu (5) Pro + Glu (305 + 127) Gln (5) Ala (5) Hpr (3) Ala + Ser + Lys (135 + 98 + 36) Gly (10) Gly (15) Gly (9) Gly (605)

Pro (5) Pro (2) Pro (74) Ser (4) Ser + Gln + Lys (57 + 41 + 11) G b (4) Pro (4) Ala (3)

a The tryptic peptides sequenced in this mixture may include the following: residues 652-654, 655-657, 652-657, and 964-974. Definitive identification of the constituent peptides would require further purification of this mixture.

and that such an influence might be exaggerated by hypergly- cemic stress. We hypothesized that the increased concentra- tion of sugar might drive the formation of glucose adducts on residues that, under normoglycemic conditions, might not respond to the relatively subtle influence of changes in higher order structure. We therefore investigated the effects of in vitro glycation on relative distribution of glucose adducts on CB3 and a2CB3-5 from._rats of different ages. In these exper- iments, intact fibers were incubated with sugar i n vitro before purification of the peptides (Fig. 1, A and B ) . The results were surprising. There were a number of significant differ- ences in glucose distribution both between young and old tissue glycated in uitro, and between tissue glycated i n uitro and tissue obtained from control animals of the corresponding age (Tables VI-IX). For example, following i n vitro glycation, Lys-479 in CB3 from 36-month-old animals had the second highest percentage of glucose adducts (27%, as compared with 2% in 36-month-old control tissue and 12% in 6-month-old tissue glycated i n uitro.) Furthermore, a stepwise increase in

relative glycation of Lys-479 could be observed at 6, 18, and 36 months (Table VIII). Relative glycation of Lys-434 was only 53% in CB3 from 36-month-old tissue that had been glycated i n uitro, as compared with 71% in CB3 from 36- month-old control tissue or with 71.5% in CB3 from 6-month- old tissue glycated in uitro. Relative glycation of Lys-453 and Lys-479 in a2C33-5 from 36-month-old tissue glycated in vitro was significantly lower than that of Lys-453 and Lys- 479 in a2CB3-5 from 36-month-old control tissue (15.5 and 14% versus 32.5 and 28.5%, respectively) (Tables VI1 and IX). Relative glycation of Lys-453 in a2CB3-5 was also lower in 36-month-old tissue glycated in uitro as compared with 6- month-old tissue glycated i n uitro. The lack of statistical significance in this latter comparison may be attributable to the closeness of the values and the low number of determi- nations (i.e. if values at 6 and 18 months are pooled, then the values at 36 months are significantly different). In summary, then, under hyperglycemic conditions i n uitro the relative distribution of glucose adducts differed in collagen from old

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24214 Sites of Nonenzymatic Glycation of Collagen i n Aging

animals as compared with collagen from young animals; dif- ferences were also observed between relative distribution of glucose adducts in collagen glycated in uitro as compared with the values in control collagen obtained from animals of the corresponding age.

Computer Analysis of Protein Structure-Several physical properties of the glycated residues in CB3 were analyzed with a computer program, based on the known primary structure of CB3 in rats. Hydrophilicity values were calculated using the Kyte-Doolittle scale. Lys-434, the predominant glycation locus, was not obviously distinguishable from the other pep- tides by any of the properties measured, nor was Lys-416, the only lysine residue with no detectable glucose adducts.

DISCUSSION

In the present study we describe an investigation of the sites of nonenzymatic glycation of two CNBr peptides from type I collagen, al(I)CB3 and a2CB3-5, prepared from tail tendon obtained from rats aged 6, 18, and 36 months. Our data show that the majority of glucose adducts are detected at only a few of the lysine residues potentially available for glycation and that such preferential glycation sites are highly conserved during aging. Under normal conditions, only minor changes in the relative distribution of glucose adducts among these sites were observed (Tables VI and VII). We also report on possible mechanisms responsible for preferential glycation of specific lysine residues.

To our knowledge, there have been few previous studies of sites of nonenzymatic glycation of collagen. Brennan (25)

TABLE VI Distribution of glucose adducts among lysine residues o n al(I)CB3

prepared from rats of different ages Data are expressed as percentages of the total glucose adducts

present on the CB3 peptide. Values for each time point represent the average of two analyses, performed on tissue obtained from two different animals. Individual values are shown in parentheses in order t o show the range of values obtained.

Age Peak (Lys residue)"

A (531) B, D (434) C (408) E (479)

months 6 18.0 (17, 19) 71 (70, 72) 1 (1, 1) 10.0 (12, 8)

18 17.4 (20, 15) 68 (64, 72) 4 (4, 4) 10.5 (12, 9) 36 23.5 (22, 25) 71 (74, 68) 3 (2, 4) 2.5 (2, 3)b

" Of the 5 lysine residues present in CB3, 4 had detectable glucose adducts. Lys-416, however, had no detectable glucose adducts and is therefore not included in this table. The peaks containing these residues, identified by letters A-E, represent the pooled fractions indicated in Fig. 5.

Significantly different from values at 6 and 18 months by AN- OVA.

recently reported on the extent of nonenzymatic glycation, as well as the content of reducible lysyl oxidase-mediated cross- links of CNBr peptides, from control and diabetic rat tail tendon collagen. We cannot directly compare our data with this study as results are reported differently and peptide preparation was different. Brennan (25) reported nonenzy- matic glycation as the fraction of total radioactivity present in CNBr peptides eluted from a polyacrylamide gel, while our data are reported as moles of glucitolyllysine per mol of peptide purified by sequential chromatography. We did find, as did Brennan (25), that virtually all of the CNBr peptides were glycated. Other studies provide indirect evidence for preferential glycation sites in collagen. Tsilibary et al. (26) found that when the NC1 domain of Type IV collagen was glycated in uitro, it was unable to bind to intact Type IV collagen, suggesting that the primary site of modification was a lysine residue close to the site mediating binding. LePape et al. (27) analyzed incorporation of ['4C]glucose by collagen extracted from normal and diabetic rat tail tendon; collagen isolated from diabetic rats incorporated considerably less radiolabeled glucose than did collagen from control rats. The investigators suggest that this decreased incorporation was due to the fact that preferential sites exist that were blocked by the presence of glucose moieties that had already bound to the collagen in vivo.

What mechanisms might be responsible for the preferential glycation of certain residues on the CB3 and a2CB3-5 pep- tides? Although this phenomenon has not previously been studied in collagen, site specificity of nonenzymatic glycation has been investigated in other proteins, including albumin, hemoglobin, low density lipoprotein, fibrin, and RNase (28- 34). In general, increased lysine reactivity with glucose is associated with relatively decreased pK, values for the t amino group of the lysine side chain (28). Shapiro et al. (31) observed that glycated lysine residues in hemoglobin were close to carboxylic acid residues in either the primary or the three- dimensional structure of the protein; he has suggested that these acidic residues contribute to local catalysis of the Ama- dori rearrangement, a reaction that favors glycation by sta- bilizing the Schiff bases. Baynes et al. (34) noted that the most reactive sites in RNase and hemoglobin were both located in high affinity binding sites for phosphate ion or organic phosphates; these sites are relatively basic regions of the molecule containing arginine and histidine residues. Baynes et al. (34) have suggested that local catalysis of the Amadori rearrangement could be influenced either by the histidine residues or by phosphate ions present in the binding site. Such an effect of the local environment could underlie increased reactivity of a specific lysine residue in cases where its pK, were not substantially lower than that of relatively

TABLE VI1 Distribution of glucose adducts among lysine residues on a2CB3-5 prepared from intact fibrils from rats of different ages

Data are expressed as percentages of the total glucose adducts present on the a2CB3-5 peptide. Values for each time point represent the average of two analyses performed on tissue obtained from two different animals. Individual values are shown in parentheses in order to show the range of values obtained.

Age Peak (Lys residue)"

A (453) E (479) c (974) D (924) E (Mixture)*

months 6 29.0 (31, 27) 23.5 (26, 21) 8.5 (5, 12) 27.5 (27, 28) 11.5 (11, 12)

18 26.5 (29, 24) 23.0 (23, 23) 13.5 (14, 13) 27.5 (25, 30) 9.5 (9, 10) 36 32.5 (34, 31) 28.5 (29, 28) 9.5 (9, 10) 21.5 (23, 20) 8 (5, 11)

' The peaks, identified by letters A-E, represent the pooled fractions indicated in Fig. 6. The tryptic peptides sequenced in this mixture may include the following: residues 652-654, 655-657, 652-657, and 964-974. Definitive

identification of the constituent peptides would require further purification of this mixture.

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Sites of Nonenzymatic Glycation of Collagen in Aging 24215

TABLE VI11 Distribution of glucose adducts among lysine residues on rul(I)CB3, from 6-month-old rats, that had been glycated i n vitro after purification;

and on CB3 prepared from intact fibrils, from rats of different ages, that had been glycated in vitro All samples shown in this table were incubated with sugar for 24 h before analysis. Data are expressed as percentages of the total glucose

adducts present on the CB3 peptide. Values for each time point represent the average of two analyses, performed on tissue obtained from two different animals. Individual values are shown in Darentheses in order to show the range of values obtained.

Sample Peak (Lys residue)"

A (531) B, D (434) C (408) E (479)

6-month-purified CB3 4.0 (3, 5)' 82.0 (84, 80)* 1.5 (1, 2) 12.5 (12, 13)' 6-month intact fibrils 13.0 (14, 12) 71.5 (73, 70) 3.5 (2, 5) 12.0 (11, 13) 18-month intact fibrils 10.5 (9, 12) 69.5 (71, 68) 4.0 (3, 5) 16.5 (18, 15)d 36-month intact fibrils 13.0 (13. 13) 53.0 (56. 50)' 2.5 (3. 2) 27.0 (28. 26)'

" The peaks containing these residues, identified by letters A-E, represent the pooled fractions indicated in Fig. 5.

' Significantly different from values for intact fibrils at 36 months by ANOVA.

I' Significantly different from values for purified CB3 and for intact fibrils at 6 and 18 months by ANOVA.

Significantly different from values for intact fibrils at all ages by ANOVA.

Significantly different from values for intact fibrils at 6 and 36 months by ANOVA.

TABLE IX Distribution of glucose adducts among lysine residues o n ru2CB3-5 prepared from intact fibrils, from rats of different ages, that had been

glycated in vitro Data are expressed as percentages of the total glucose adducts present on the a2CB3-5 peptide. Values for each time point represent the

average of two analyses performed on tissue obtained from two different animals. Individual values are shown in parentheses in order to show the range of values obtained.

Age Peak (Lys residue)"

A (453) B (479) c (974) D (924) E (mixture)

months 6 26 (27, 25) 12 (15, 9) 16.5 (16, 17) 34 (31,371 11.5 (11, 12)

15.5 (20. 11) 15 (15. 15) 22.5 (18. 27) 34.5 (36. 33) 12.5 (11. 14) 18 26 (23, 29) 14 (14, 14) 18.5 (20, 17) 33.5 (37, 30) 36

8.0 (6, 10)

"The peaks, indicated by letters A-E, correspond to the peaks shown in Fig. 6.

nonreactive residues. Several of these proposed mechanisms may be operative in collagen. For example, the predominant glycation locus in CB3 is Lys-434, the only lysine immediately adjacent to an acidic residue (aspartic acid at position 435). In addition, aspartic acid is present in the analogous position in the a2 chain at residue 435. Perhaps these structural features are conducive to the formation of a cationic pocket in the tropocollagen molecule that is capable of binding buffer anions or ligands that may catalyze the Amadori re- arrangement (35). Similarly, Lys-924 in the a2CB3-5 chain is adjacent to an Asp residue (position 923 in the a2 chain) as well as 3 Asp residues at position 926 in all three of the a chains. The microenvironment of Lys-453 in a2CB3-5 may be conducive to glycation due to the proximity of arginine (position 453 in the al(1) chain) and glutamic acid (position 452 in both the al(1) and a2 chains, as well as at position 455 in all three chains). The third major glycation site in a2CB3- 5, Lys-479, has no such structural features to account for preferential glycation of this residue; however, it should be noted that the rat a2 chain has not as yet been sequenced in this region and that it is possible that there are key differences in 1 or 2 residues adjacent to lysine in the rat sequence.

Our initial observations concerning site specificity of non- enzymatic glycation had been made in collagen obtained from young animals (9). We were surprised to find in the present study that the preferential glycation sites remained virtually unchanged even in extremely old rats (Tables VI and VII), despite the profound structural changes collagen undergoes with aging. These results suggested that primary structure of collagen may be a major determinant of the location of adduct formation. To explore this possibility further, we investigated the relative influence of primary structure and of higher order structure on glycation sites by incubating purified CB3 and intact fibrils in vitro with sugar, and then analyzing glycation

sites on CB3. If higher order structure was a necessary deter- minant of preferential glycation sites, then we would predict that sugar adducts would be more randomly distributed among the 5 lysine residues in purified CB3 incubated with sugar in vitro than in intact fibrils incubated i n vitro. Although some sugars were already present on the peptide (as we were unable to separate glycated CNBr peptides from nonglycated pep- tides), we knew from previous experiments that incubation in vitro would increase moles of glucose per mol of collagen at least 2-3-fold, thus allowing us to easily appreciate any in- crease in randomness of distribution. The results (Table VIII) clearly show that far from increasing the randomness of glycation sites, purification of CB3 before glycation resulted in increased preference for the predominant glycation site (Lys-434). Since purified CB3 presumably exists as a random coil in the glucose solution, while intact fibers retain their triple helical conformation, it seems unlikely that higher order structure contributes significantly to the predilection of Lys- 434 to form adducts with sugar. However, higher order struc- ture may influence the relative distribution of glucose adducts among the preferred sites.

We wondered if the more subtle effects of higher order structure, as compared with primary structure, on glycation sites might be revealed by perturbations of normal conditions. To explore this possibility, we incubated intact collagen fibrils from 6-, 18-, and 36-month-old animals with sugar and ana- lyzed glycation sites in both CB3 and a2CB3-5. We found that distribution of glucose adducts was not the same in old collagen as in young collagen under hyperglycemic conditions in vitro or between collagen glycated i n vitro and control collagen from animals of the same age. It is certainly possible that the changes in higher order structure that are associated with aging have the capacity to affect glucose adduct forma- tion. For example, morphologic studies indicate that there is

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24216 Sites of Nonenzymatic Glycation of Collagen in Aging

a loss of order in the fibrillar array, including shortening and thinning of the collagen bundles, increased irregularity and roughness, and increased interfibrillar space, corresponding t o ground substance (6). The causes of these changes are not known, but may include distortion of collagen arrays by the accumulation of advanced glycation products as well as alter- ations in the interactions between collagen and other macro- molecules in the matrix, particularly fibronectin (1, 6, 7). Such structural changes may well influence the microenviron- ment of selected lysine and hydroxylysine residues on the collagen molecule and hence their predilection to form ad- ducts with sugar. Although under normal conditions supras- tructural changes may be less influential than, for example, is primary structure (hence the conservation of preferential glycation sites in old collagen), perturbations such as hyper- glycemic stress may permit these suprastructural effects to be appreciated. An additional consideration, a t least with regard t o a2CB3-5, may be the influence of age-associated changes in enzyme-mediated cross-linking on nonenzymatic glycation.

What are the implications of our observations concerning preferential glycation sites in collagen and their conservation with age? Although it has been observed that changes in nonenzymatic glycation may affect many properties of colla- gen, including ligand binding, conformation, and molecular packing (1,26,36), it is not yet known if there is a relationship between glycation sites and effects on specific physicochemi- cal properties. Of particular interest, especially in terms of aging, is the relationship between sites of glucose adduct formation and the location of advanced Maillard products. These advanced glycation products increase with age in all tissues that have been studied and are known to have pro- found effects on many physicochemical properties of collagen. I t is not yet known if they accumulate at all major glycation sites or if they also have preferential sites of formation.

The conservation of preferential glycation sites on collagen during aging has both theoretical and practical implications. We initially hypothesized that the highly specific location of glucose adducts observed in young animals would change with age to become more random as structural order decreased. In other words, we hypothesized that the information necessary t o direct preferential glycation would be increasingly unavail- able as the fibrillar arrays became more disorganized. We were particularly interested in determining if a relationship existed between loss of structural complexity and a specific biochemical event (nonenzymatic glycation) in light of recent suggestions by Lipsitz and Goldberger (37, 38) that loss of complexity, as measured by fractal analysis, may provide an index of aging. We postulated that increasing structural dis- order would lead to increased randomness of glucose adduct formation, which would result in increasingly random location of advanced glycation products, which, in turn, would further increase the disorder in the fibrillar arrays; in other words, a feedback loop. However, contrary to our hypothesis, we found little change in preferential glycation sites with aging under normoglycemic conditions. Our data strongly suggest that the information present in primary structure alone is sufficient to maintain order during the first step in nonenzymatic gly- cation of collagen: the formation of glucose adducts on specific residues. From the standpoint of developing interventional strategies, then, our findings suggest that one does not have

to intervene in a feedback loop that generates increased randomness at each iteration. Rather, it may be possible to prevent some of the deleterious changes that occur in connec- tive tissue with aging by blocking glucose adduct formation at specific loci and/or preventing the formation of advanced glycation products at specific loci. Possible approaches might include blocking the reactive residues, blocking adjacent res- idues so as to decrease the favorable milieu for adduct for- mation, preventing the formation of advanced glycation prod- ucts (with agents such as aminoguanidine), and perhaps even introducing alterations in the primary structure of collagen. Further elucidation of the relationship between early and advanced glycation products and the physicochemical prop- erties of collagen as they change with age may suggest addi- tional interventional strategies.

Acknowledgments-We thank Judith Shimizu and Susan DeSautel for skilled technical assistance.

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