o-glycosylation of insulin-like growth factor (igf) binding protein-6 maintains high igf-ii binding...

O-glycosylation of insulin-like growth factor (IGF) binding protein-6maintains high IGF-II binding affinity by decreasing binding toglycosaminoglycans and susceptibility to proteolysis

Joe A. Marinaro1, Gregory M. Neumann2, Vincenzo C. Russo3, Kerri S. Leeding1 and Leon A. Bach1

1University of Melbourne, Department of Medicine, Austin & Repatriation Medical Centre, Heidelberg, Victoria, Australia; 2La Trobe University,

Department of Biochemistry, Bundoora, Victoria, Australia; 3Centre for Hormone Research, Royal Children's Hospital, Parkville, Victoria,

Australia

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits

insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycos-

aminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied.

Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was

approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans,

IGFBP-6 had < 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6

also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited

by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also

protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding

affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed

with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a

4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding

site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In

contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide

which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to

glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-

affinity, soluble form and so contributing to its inhibition of IGF-II actions.

Keywords: binding protein; glycosylation; insulin-like growth factor; glycosaminoglycan; proteolysis.

The insulin-like growth factors, IGF-I and IGF-II, stimulategrowth and differentiation of many cell types [1]. Actions of theIGFs are modulated by a family of six high-affinity IGF bindingproteins (IGFBPs) [1,2]. IGFBPs are expressed by most, if notall, cell types. Individual IGFBPs differ in their bindingaffinities for IGFs, tissue distribution, regulation and post-translational modifications.

IGFBPs may inhibit or potentiate IGF actions. The mechan-ism underlying inhibition of IGF actions is thought to besequestration of free IGFs and the consequent prevention ofbinding to IGF receptors. In contrast, the mechanisms under-lying potentiation of IGF actions are not completely understood[1,2]. Some IGFBPs interact with cell membranes and/oradjacent extracellular matrix, which may be an important factorfor potentiation of IGF actions [1,2]. IGFBP-2, IGFBP-3 andIGFBP-5 bind to cell membrane proteins and/or proteoglycans,

and their sequences contain highly basic putative heparinrecognition sequences that are responsible for glycosamino-glycan binding [3±6]. When associated with cells or bound toglycosaminoglycans, the IGF binding affinities of theseIGFBPs are reduced, and it is thought that this allows releaseof IGFs for binding to IGF receptors, thereby resulting inpotentiation of IGF actions.

Recent evidence suggests that proteolysis of IGFBPs alsomodulates their effects on IGF actions by decreasing theirbinding affinities for IGFs [7]. This may be another factorcontributing to potentiation of IGF actions. However, there havebeen few detailed studies of the structural basis of the decreasein IGF binding affinity following IGFBP proteolysis.

IGF-II is a potent mitogen, the expression of which isincreased in many tumours [8]. IGFBP-6 is a relativelyspecific inhibitor of IGF-II actions [9], at least in part due to its20±100-fold higher binding affinity for IGF-II over IGF-I[10±14]. Expression of IGFBP-6 is associated with nonpro-liferative states such as differentiating and quiescent cells [9].Differentiating agents such as retinoic acid increase IGFBP-6levels in cells [15], and overexpression of IGFBP-6 inhibitsxenografted neuroblastoma growth in vivo [16]. These resultssuggest that IGFBP-6 is a potent antiproliferative protein, mostlikely due to inhibition of IGF-II actions.

Similarly to IGFBP-3 and IGFBP-5, IGFBP-6 has a highlybasic amino acid region containing a putative heparin binding

Eur. J. Biochem. 267, 5378±5386 (2000) q FEBS 2000

Correspondence to L. Bach, University of Melbourne, Department of

Medicine, Austin & Repatriation Medical Centre (Austin Campus), Studley

Road, Heidelberg, Victoria, 3084, Australia. Fax: 161 39457 5485,

Tel.: 161 39496 3581, E-mail: [email protected]

Abbreviations: g, glycosylated; IGF, insulin-like growth factor;

IGFBP, insulin-like growth factor binding protein; n-g,

nonglycosylated; TCEP, tris(carboxyethyl)phosphine; AEBSF,

4-(2-aminoethyl)benzene]sulphonyl-fluoride.

(Received 22 March 2000, revised 12 June 2000, accepted 19 June 2000)

site, but IGFBP-6 is cell-associated only to a very limited extentin some cells and not at all in others [4,17,18]. However, peptidescorresponding to the heparin binding sequence of IGFBP-6 arecapable of binding to cells [4,19], suggesting that other factorsmay preclude cell association of full-length IGFBP-6.

IGFBP-6 is O-glycosylated [20,21] but the effects of glyco-sylation on its properties are incompletely understood.O-glycosylation delays the clearance of IGFBP-6 from thecirculation [22], but it does not directly influence high affinityIGF binding by IGFBP-6 [20,23]. However, its IGF bindingaffinity may be decreased by cell association or proteolysis, andboth of these processes may be inhibited by O-glycosylation asglycosylation of other proteins inhibits their interactions withglycosaminoglycans [24,25] or susceptibility to proteolysis[26]. One aim of this study was therefore to determine the effectof O-glycosylation on the binding of human IGFBP-6 toglycosaminoglycans and cell membranes. Our previous studieshave shown that O-glycosylation inhibits proteolysis ofIGFBP-6 by trypsin and chymotrypsin [21]. We therefore alsoinvestigated the effect of IGFBP-6 proteolysis on IGF-IIbinding and the structural basis of the reduced IGF bindingaffinity of IGFBP-6 following cleavage by these proteases. Inthese studies a comparison was made between recombinanthuman IGFBP-6 expressed in Chinese hamster ovary cells,which is glycosylated to a similar extent to native IGFBP-6, andnonglycosylated IGFBP-6 expressed in Escherichia coli[17,21].

M A T E R I A L S A N D M E T H O D S

Materials

Immunolon-4 microtitre immunoassay plates were purchasedfrom Dynatech Laboratories Inc. Aggrecan was kindly pro-vided by A. Fosang (Royal Children's Hospital, Melbourne,Australia). Purified keratan sulfate and glycosaminoglycansenriched in chondroitin-4 sulfate and chondroitin-6 sulfatewere a kind gift of D. Lowther (Monash University,Australia). Chondroitin ABC lyase and keratanase were fromSeikagaku Kogyo (Japan). E-64 and pepstatin were fromBoehringer-Mannheim. [4-(2-aminoethyl)benzene]sulphonyl-fluoride (AEBSF) was from Calbiochem-Novabiochem. PC12rat phaeochromocytoma cells were provided by D. Bowtell(Peter MacCallum Cancer Institute, Melbourne, Australia).Phenylmethanesulfonyl fluoride, disuccinimidyl suberate,Tris(carboxyethyl)phosphine (TCEP) and BSA (RIA grade)were from Sigma.

Expression and purification of recombinant glycosylated andnonglycosylated IGFBP-6

Recombinant human glycosylated IGFBP-6 (gIGFBP-6) wasexpressed and purified from Chinese hamster ovary cells asdescribed previously [21]. The IGFBP-6 used for experi-ments was . 99% pure as determined by N-terminal Edmansequencing. Recombinant human nonglycosylated IGFBP-6(n-gIGFBP-6) was expressed and purified from E. coli asdescribed previously [21]. As anticipated, IGFBP-6 synthesizedby E. coli was not glycosylated as confirmed by the mass of22737.6 ^ 3.3 Da measured by ESI-MS compared with acalculated mass of 22735.1 Da. n-gIGFBP-6 had similar high-affinity IGF binding characteristics as gIGFBP-6, and the twopreparations had similar specific activities [23]. As high affinitybinding is dependent on correct folding, these results indicatethat n-gIGFBP-6 is folded correctly.

Binding of IGFBP-6 to aggrecan and glycosaminoglycans

Immulon-4 96-well plates were coated with 500 ng aggrecan orglycosaminoglycans (chondroitin-4 sulfate, chondroitin-6 sul-fate, keratan sulfate or heparan sulfate) in 200 mL NaCl/Pi for16 h at 37 8C. Excess salt and unbound reagents were removedby washing and wells were blocked with 1% BSA. Wells werethen washed and incubated in binding buffer (16 mm Tris/HClpH 7.2, 50 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 1 mg´mL21

BSA, 0.02% Tween-20) with or without 10 ng of gIGFBP-6 orn-gIGFBP-6. To determine whether IGFBP-6 bound to theglycosaminoglycan component of aggrecan, binding was alsomeasured to aggrecan previously digested with chondroitinaseABC or keratanase (0.25 U´mg21) in the presence of proteaseinhibitors (10 mm EDTA, 20 mg´mL21 E-64, 1 mm AEBSF and2 mm pepstatin) at 37 8C for 3 h.

Following removal of unbound IGFBPs by washing,glycosaminoglycan-bound IGFBP-6 was detected by incuba-tion with [125I]IGF-II (3 � 104 c.p.m.´well21, specific activity105 mCi´mg21). Wells were washed, bound radioactivity wassolubilized with 200 mm NaOH, 0.1% Triton X-100, and[125I]IGF-II binding was quantitated in a g-counter. Each pointwas measured in triplicate within each experiment. Specificitywas determined by measuring binding in: (a) wells coated withglycosaminoglycans without added IGFBP-6; and (b) wellscoated with BSA with or without IGFBP-6. Specificity of[125I]IGF-II binding was determined by coincubation withexcess unlabelled IGF-II (13.4 nm).

Binding affinity of glycosaminoglycan-bound and solubleIGFBP-6 for IGF-II

Microtiter 96-well plates were coated with chondroitin-6sulfate and incubated with gIGFBP-6 or n-gIGFBP-6 (20 ng)as described above. Unbound IGFBPs were removed and wellswere incubated with [125I]IGF-II (3 � 104 c.p.m./well) in thepresence of unlabelled IGF-II (0±13.4 nm). Wells were washedand bound radioactivity was measured in a g-counter. Non-specific binding (2.6 ^ 0.7%) was assessed by measuringbinding in the absence of added IGFBP. Specific binding wasdetermined by subtraction of nonspecific from total binding.This experiment was performed three times, with each pointmeasured in triplicate in each experiment. Affinity constantswere determined using ligand [27].

The IGF-II binding of n-gIGFBP-6 and gIGFBP-6 in solutionwas measured by competitive binding assay as described pre-viously [28]. Following incubation of IGFBP and [125I]IGF-IIin solution, bound and free IGFs were separated using albumin-coated charcoal followed by centrifugation at 1300 g. Radio-activity in the supernatant was counted. Nonspecific binding(3.8 ^ 0.2%) was assessed by measuring adsorption of tracerto charcoal in the absence of added IGFBP. Specific bindingwas determined by subtraction of nonspecific from totalbinding. This experiment was performed three times, witheach point measured in duplicate within each experiment.Affinity constants were determined using ligand [27].

PC12 membrane preparation

Confluent PC12 cells from a 75-cm2 flask were resuspended inice-cold buffer (10 mm Tris/HCl pH 7.4, 2 mm phenylmethane-sulfonyl fluoride, 1 TIU´mL21 aprotinin) and mechanicallydisaggregated through 19-gauge and 23-gauge needles. The cellsuspension was centrifuged at 500 r.p.m. for 5 min at 4 8C. Thesupernatant was then centrifuged at 14 000 r.p.m. for 1 h at

q FEBS 2000 O-glycans inhibit glycosaminoglycan binding and proteolysis of IGFBP-6 (Eur. J. Biochem. 267) 5379

4 8C. The membrane pellet was resuspended in ice-cold 10 mmTris/HCl pH 7.4 and stored at 220 8C. The protein content ofPC12 membranes purified from one flask was < 800 mg.

Affinity cross-linking

PC12 membranes (100 mg´50 mL21) were incubated for 1 h at4 8C with or without gIGFBP-6 or n-gIGFBP-6 (20 ng).Samples were then centrifuged at 14 000 r.p.m. for 20 min at4 8C. The pellet was washed to remove unbound IGFBP-6,re-centrifuged and then incubated with [125I]IGF-II (5 � 104

c.p.m.) for 2 h at 37 8C. Nonspecific binding was determinedby coincubation with excess unlabelled IGF-II (27 nm).Samples were placed on ice for 10 min before cross-linkingwas performed with disuccinimidyl suberate (final concentra-tion, 1 mm) for 15 min at 4 8C. Cross-linking was terminatedwith 0.1 m Tris/HCl pH 7.4, 20 mm EDTA on ice for 5 min.Samples were separated by SDS/15% PAGE, dried and exposedto X-ray film for 1±7 days.

To determine the effects of ionic strength on IGFBP-6binding to PC12 membranes, membranes were incubated withIGFBP-6 in the presence of NaCl (0.15±0.5 m) for 1 h at 37 8Cfollowed by cross-linking as described above.

Western ligand blotting

Proteins from PC12 membrane extracts and conditioned mediawere separated by SDS/15% PAGE and transferred to anitrocellulose membrane. The membrane was blocked with1% BSA, probed with [125I]IGF-II (1 � 106 c.p.m.), washedand exposed to X-ray film (Biomax, Kodak, Coburg, Australia)for 24 h.

Limited proteolysis of IGFBP-6 with trypsin or chymotrypsin

gIGFBP-6 or n-gIGFBP-6 (200 ng) were incubated with0±10 ng of trypsin or chymotrypsin for 2 h at 37 8C in100 mm NaCl, 50 mm Tris/HCl (pH 8.0). Digestions wereterminated by boiling in nonreducing Laemmli sample bufferfor 10 min. Following incubation with proteases, IGFBP-6 wasanalysed by SDS/PAGE followed by immunoblotting using ahuman IGFBP-6 polyclonal antiserum (kindly provided by J.Martin, Kolling Institute, St Leonards, Australia) [29] andWestern ligand blotting as described above. Additionally,gIGFBP-6 (200 ng) was incubated with chymotrypsin asindicated above and digestions terminated by boiling. Samplevolumes corresponding to 0.01±10 ng of IGFBP-6 were thenassayed in solution.

ESI-MS

gIGFBP-6 (10 mg) was digested with chymotrypsin (100 ng)in 25 mm ammonium bicarbonate (pH 8) for 2 h at 37 8C.Digestion was stopped by boiling for 5 min followed byacidification to pH 2 with 0.1% formic acid. Half of the digestwas subjected to disulphide bond reduction using 100 mmTCEP (50 8C, 60 min), followed by reversed phase desaltingwith a C18 micropipette tip (Millipore ZipTip, 0.1% formicacid wash, 50% acetonitrile/0.1% formic acid elution). Sampleswere analysed by ESI-MS as described previously [21], using aPerkin-Elmer Sciex API-300 triple quadrupole mass spec-trometer fitted with a microionspray ion source (flow rate0.2 mL´min21). Singly charged poly(propylene glycol) ionswere used to calibrate the Q1 m/z scale (30±3000 Da perunit charge). PE-Sciex BioMultiView software was used to

transform signal-averaged mass spectra to a true mass scale andto analyse ms/ms collisional fragmentation spectra (Q3 scans)obtained as described previously [30].

Statistics

Results are shown as mean ^ SEM of three to five independentexperiments. Within each experiment, points were measured induplicate or triplicate. Binding of gIGFBP-6 and n-gIGFBP-6to glycosaminoglycans was analysed by one-way analysis ofvariance. IGF-II binding affinities of soluble and glysosamino-glycan-bound IGFBP-6 were compared by analysis of varianceafter log transformation to stabilize variance. Uncertainties inESI-MS masses (typically ^ 0.01%) were calculated as 95%confidence limits from small sample statistics includingcalibration uncertainty.

R E S U LT S

O-glycosylation inhibits binding of IGFBP-6 toglycosaminoglycans

Following incubation of n-gIGFBP-6 and gIGFBP-6 in wellscoated with a range of glycosaminoglycans, binding of[125I]IGF-II was approximately threefold higher with non-glycosylated (25.5±30.1% of added radioactivity) than withglycosylated IGFBP-6 (9.2±11.7% of added radioactivity,Fig. 1). This assay, which has been used previously to showbinding of IGFBP-2 to glycosaminoglycans [6], was validatedusing the following controls. When gIGFBP-6 or n-gIGFBP-6were incubated in wells coated with BSA, a control protein,[125I]IGF-II binding was only 2.0 ^ 1.0% of added radio-activity (Fig. 1). In the absence of added IGFBP-6, only2.0 ^ 0.4% of added [125I]IGF-II bound to glycosamino-glycans (results not shown), indicating that [125I]IGF-II bindingrequires the presence of IGFBP-6 in the assay. Co-incubation of

Fig. 1. O-Glycosylation inhibits binding of IGFBP-6 to glycosamino-

glycans. Microtitre 96-well plates were coated with aggrecan (Agg),

chondroitin-4 sulfate (C-4S), chondroitin-6 sulfate (C-6S), keratan sulfate

(KS) or heparan sulfate (HS) (500 ng´mL21). Ten ng of n-gIGFBP-6 (open

bars) or gIGFBP-6 (filled bars) were incubated for 1 h at 37 8C. Wells were

washed and incubated with [125I]IGF-II (3 � 104 c.p.m. per well) for 16 h

at 4 8C. Wells coated with BSA were incubated in the same way and used as

controls. Following incubation, wells were washed and bound radioactivity

was solubilized and measured in a g-counter. Results are shown as mean

^SEM of three experiments, within each point measured in triplicate

within each experiment. *P , 0.05; **P , 0.01.

5380 J. A. Marinaro et al. (Eur. J. Biochem. 267) q FEBS 2000

[125I]IGF-II with excess unlabelled IGF-II (13.4 nm) reducedbinding to 1.0 ^ 0.8% (results not shown), indicating that thetracer binding to IGFBP-6 was specific.

As binding of other IGFBPs to cell membranes, extracellularmatrix and/or glycosaminoglycans decreases their IGF bindingaffinities [3,6,31,32], the IGF-II binding affinities of chon-droitin 6-sulfate-bound and soluble n-gIGFBP-6 were com-pared. Affinity constants for soluble and bound n-gIGFBP-6were 3.6 ^ 1.1 � 10211 m and 2.8 ^ 1.8 � 10210 m, respec-tively. Although the amount of IGF-II binding to gIGFBP-6bound to chondroitin 6-sulfate was less than that of n-gIGFBP-6,there was a similar reduction in IGF-II binding affinity (Kd

soluble 1.9 ^ 0.6 � 10211 m vs. bound 2.1 ^ 0.5 � 10210 m).Binding of IGFBP-6 to chondroitin 6-sulphate therefore resultsin an < 10-fold decrease in IGF-II binding affinity (P � 0.0007).In contrast, glycosylation has no effect on the IGF-II bindingaffinity of IGFBP-6 in solution or when immobilized by bindingto chondroitin 6-sulphate (P � 0.78). The higher [125I]IGF-IIbinding following incubation of glycosaminoglycans withn-gIGFBP-6 than gIGFBP-6 (Fig. 1) therefore reflects inhi-bition of IGFBP-6 binding to glycosaminoglycans byO-glycosylation.

To determine whether IGFBP-6 could bind to glycosamino-glycans within a proteoglycan, experiments using aggrecan(which contains chondroitin sulfate and keratan sulfateglycosaminoglycans) were performed. Although the absolutelevel of IGFBP-6 binding to aggrecan was lower than those ofbinding to glycosaminoglycans, binding of n-gIGFBP-6 toaggrecan (10.2 ^ 1.6% of added radioactivity) remained2.5-fold higher than gIGFBP-6 (4.0 ^ 0.1% of added radio-activity, Fig. 1, Agg). Digestion of aggrecan with enzymeswhich digest specific glycosaminoglycans (either chondroiti-nase ABC or keratanase) each decreased gIGFBP-6 andn-gIGFBP-6 binding to aggrecan by < 50% (results notshown), confirming that IGFBP-6 is binding to the glycosamino-glycan side chains of this proteoglycan.

O-glycosylation inhibits binding of exogenous IGFBP-6 toPC12 cell membranes

Cell-membrane association of IGFBP-6 has not been widelyobserved. However, we have shown that a small amount of ratIGFBP-6 may be found in PC12 rat phaeochromocytoma celllysates, possibly reflecting cell-membrane binding [18]. In thepresent study, small amounts of rat IGFBP-6 were identified ina PC12 membrane preparation by Western ligand blotting andimmunoblotting (results not shown). Incubation of membraneswith 0.5 m NaCl resulted in elution of IGFBP-6 (results notshown), consistent with disruption of ionic interactions such asthat seen in binding of proteins to glycosaminoglycans.

Since the above studies showed that PC12 membranes canbind rat IGFBP-6, they were used to study the role ofO-glycosylation on cell membrane binding of human IGFBP-6.Affinity cross-linking of PC12 membranes with [125I]IGF-IIrevealed a 30-kDa IGF/IGFBP complex (Fig. 2, lane 2), whichis consistent with rat IGFBP-6 as described above. Excessunlabelled IGF-II abolished the 30 kDa signal (Fig. 2, lane 1),confirming specificity. Following incubation with n-gIGFBP-6,the 30-kDa band identified by affinity cross-linking increasedin intensity (Fig. 2, lane 4). As n-gIGFBP-6 and [125I]IGF-IIhave molecular weights of < 23 kDa and 7.5 kDa, respectively,this is consistent with the presence of membrane-boundn-gIGFBP-6. In contrast, incubation of gIGFBP-6 with PC12membranes did not result in the appearance of an < 38 kDacomplex consisting of < 30 kDa human gIGFBP-6 and

[125I]IGF-II (Fig. 2, lane 3). Incubation of n-gIGFBP-6 withPC12 membranes in the presence of NaCl (0.15 m or 0.5 m)decreased binding (Fig. 2, lanes 5±7), consistent withn-gIGFBP-6 binding to PC12 membranes via interactionswith glycosaminoglycans.

O-glycosylation inhibits proteolysis of IGFBP-6

Chymotrypsin. Prostate-specific antigen, which has chymo-trypsin-like activity, cleaves IGFBP-3 [33], and it had beenshown previously that O-glycosylation inhibits digestion ofIGFBP-6 by chymotrypsin [21]. The structural and functionalconsequences of chymotrypsin digestion of IGFBP-6 weretherefore studied.

Inhibition of chymotrypsin digestion of IGFBP-6 byO-glycosylation was confirmed in the present study (Fig. 3A).Chymotrypsin reduced the apparent molecular mass of bothgIGFBP-6 and n-gIGFBP-6 by < 3±5 kDa as determined byimmunoblotting, but considerably more digestion was observedin the absence of glycosylation (Fig. 3A). For example,following digestion with 1 ng chymotrypsin, the major fragmentof n-gIGFBP-6 and intact n-gIGFBP-6 had equal intensities,whereas the major gIGFBP-6 fragment was less intense thanintact gIGFBP-6 (Fig. 3A). The same membranes were used toanalyse IGF-II binding by Western ligand blotting (Fig. 3B).Following cleavage with increasing amounts of chymotrypsin,IGF-II binding decreased progressively. In particular, fragmentsof gIGFBP-6 or n-gIGFBP-6 did not bind [125I]IGF-II. AsO-glycosylation inhibited digestion of IGFBP-6 by chymo-trypsin, the loss of IGF-II binding was substantially less withgIGFBP-6 compared with n-gIGFBP-6. Solution binding assaysconfirmed the decrease in binding of IGF-II resulting fromdigestion of IGFBP-6 by chymotrypsin (results not shown).

It is intriguing that a relatively small (3±5 kDa) reduction inapparent molecular mass due to incomplete chymotrypticdigestion of gIGFBP-6 (Fig. 3A) results in the complete lossof IGF-II binding (Fig. 3B). To investigate the specific peptidebond cleavages responsible for this loss of IGF binding,

Fig. 2. Exogenous n-gIGFBP-6 binds to PC12 rat pheochromocytoma

membranes. n-gIGFBP-6 and gIGFBP-6 were incubated with PC12

membranes for 1 h at 37 8C. Following washing to remove unbound

IGFBPs, membranes were incubated with [125I]IGF-II (5 � 104 c.p.m./

well) in the presence (lane 1) or absence (lanes 2±4) of unlabelled IGF-II

(27 nm). n-gIGFBP-6 was also incubated with membranes in the presence

of NaCl (lanes 5±7). After affinity cross-linking, autoradiography was

performed for 1 day (lanes 1±4) or 4 days (lanes 5±7). Migration of

reduced molecular mass markers is shown on the left. This figure is

representative of three independent experiments. Lane 1, Nonspecific

binding; lane 2, membranes with no added IGFBPs; lane 3, membranes 1

gIGFBP-6; lane 4, membranes 1 n-gIGFBP-6; lanes 5±7, membranes

1 n-gIGFBP-6; lane 5, 0 m NaCl; lane 6, 0.15 m NaCl; lane 7, 0.5 m NaCl.


incomplete chymotryptic digests of gIGFBP-6 were analysed byESI-MS (Fig. 4, Table 1). All disulphide bonds in an incompletechymotryptic digest of gIGFBP-6 were reduced using TCEP,followed by ESI-MS analysis and ms/ms collisional fragmen-tation for increased certainty of identification. Peptidesencompassing all of IGFBP-6 except the glycosylated regionLeu99±Leu167 were identified (Table 1) and indicate that

Fig. 4. ESI-MS of chymotrypsin-digested glycosylated IGFBP-6. Sam-

ples (5 mg) of gIGFBP-6 were digested with chymotrypsin (50 ng) for 2 h

at 37 8C and analysed by ESI-MS. Raw mass spectra (not shown) were

analysed manually before computerized transformation to a true mass scale

(as shown). Two prominent higher mass components were present, with

related minor peaks identifying these components as: A, 4551 Da C-terminal

peptide fragment; and B, 11388 Da N-terminal glycopeptide fragment (see

Table 1). NeuAc is N-acetylneuraminic acid (sialic acid).

Table 1. ESI-MS analysis of gIGFBP-6 partially digested by chymotrypsin. Aliquots (5 mg) of gIGFBP-6 were digested with chymotrypsin (50 ng) for

2 h at 37 8C and disulphide bonds either (A) reduced using 100 mm TCEP (50 8C, 60 min) or (B) not reduced, followed by ESI-MS analysis (Fig. 4).

Calculated masses based on the IGFBP-6 sequence (Fig. 5) are average molecular masses above 2000 Da and monoisotopic below 2000 Da. Peptide

sequences of fragments with masses less than 2500 Da were confirmed by ms/ms collisional fragmentation (results not shown).

Calculated mass (Da) Observed mass (Da) Difference (Da)

Reduced fragments

(Arg28±Tyr74) 4662�.1 4661.5 �̂ 0.7 2 0.6 �^ 0.7

T75PNCAPGLQCHPPKDDEAPL94 2103�.4 2103.0 �^ 0.6 2 0.4 �^ 0.5

R95ALL98 471�.3 471.5 �^ 0.3 1 0.2 �^ 0.3

(Leu99±Leu167)a 9�±11 kDa Not det�ected

(D168SVLQQLQTEVY179)b 1421�.7 1421.8 �^ 0.4 1 0.1 �^ 0.4

(Q172QLQTEVY179)b 1007�.5 1007.6 �^ 0.4 1 0.1 �^ 0.4

R180GAQTLY186 807�.4 807.6 �^ 0.3 1 0.2 �^ 0.3

V187PNCDHRGF195 1043�.5 1043.6 �^ 0.4 1 0.1 �^ 0.4

V187PNCDHRGFY196 1206�.5 1206.8 �^ 0.5 1 0.3 �^ 0.5

(Arg197±Ser239) 4555�.1 4555.2 �̂ 0.7 1 0.1 �^ 0.7

Unreduced fragments

(Arg28±Tyr74)(Thr75±Leu94)(Leu98±Val132)c 10439�.6 11387.6 �^ 1.4 1 948.0 �^ 1.4

R95AL97 357�.5 Not de�tected

(Asn133±Tyr179)(Val187±Tyr196)d 7�±9 kDa Not de�tected

R180GAQTLY186 807�.4 807.6 �^ 0.3 1 0.2 �^ 0.3

(Arg197-Ser239)e 4551�.1 4550.8 �^ 0.6 2 0.3 �^ 0.6

aHeterogeneously glycosylated fragment [21]. bMinor peaks. cDisulphide-linked complex glycosylated on Thr126 with a 947.9-Da oligosaccharide

(composition N-acetylhexosamine, hexose and two N-acetylneuraminic acids) [21]. This assignment is supported by a minor peak due to additional

N-terminal Leu26±Arg as observed previously [21,30], and a peak 291 Da lower in mass, characteristic of loss of N-acetylneuraminic acid [21] (Fig. 4B). A

smaller peak corresponding to loss of leucine (Fig. 4B), consistent with partial digestion after Leu98 in addition to Leu97, was also observed.dHeterogeneously glycosylated disulphide-linked complex containing one S±S bond [21]. eAssignment supported by minor peaks (Fig. 4A) due to an

additional C-terminal Gly240 as observed previously [21] and an additional Tyr196 due to cleavage after Phe195 instead of Tyr196.

Fig. 3. O-glycosylation protects IGFBP-6 from digestion by chymo-

trypsin. Glycosylated (G) and nonglycosylated (N) IGFBP-6 (200 ng) were

digested with chymotrypsin for 2 h at 37 8C. Samples were analysed by

SDS/PAGE followed by (A) immunoblotting and (B) Western ligand

blotting using [125I]IGF-II on the same membranes. This figure is represen-

tative of three independent experiments.


chymotrypsin preferentially cleaves gIGFBP-6 after Tyr74,Leu94, Leu98, Tyr179, Tyr186 and Phe195/Tyr196 (numberingbased on IBP6_HUMAN, SWISS-PROT accession numberP24592, Fig. 5).

ESI-MS analysis under nonreducing conditions revealedthree main components with masses of 4551, 807.4 and11388 Da (Table 1). The prominent 4551-Da componentcorresponds to the C-terminal fragment Arg197±Ser239released by cleavage after Tyr196 (Figs 4A and 5). The807.6-Da fragment present in both nonreduced and reduceddigests was identified as R180GAQTLY186 (Table 1). The11388-Da component (Fig. 4B, Table 1) was identified as aglycosylated N-terminal fragment containing only one of thefive previously determined glycosylation sites [21], namelyThr126, which carries a 947.9-Da tetrasaccharide (Table 1).This fragment corresponds to a disulphide-linked complexconsisting of Arg28±Tyr74, Thr75±Leu94 and Leu98±Val132(Fig. 5). The inferred cleavages after Tyr and Leu are typical

for chymotrypsin [34], but cleavage after Val132 was notexpected and this may not represent a major chymotrypticcleavage site. Indeed, digestion of gIGFBP-6 at all inferredmajor chymotryptic cleavage sites (Tyr74, Leu94, Leu97/Leu98, Tyr179, Tyr186 and Phe195/Tyr196), but not afterVal132, would generate an < 19±21-kDa fragment consistentwith that observed by SDS/PAGE (Fig. 3A).

From the ESI-MS data, it is concluded that the < 20-kDachymotryptic gIGFBP-6 fragment observed on nonreducingSDS/PAGE (Fig. 3A) probably consists of gIGFBP-6 which haslost C-terminal Tyr196/Arg197-Ser239 and two excised peptides,Arg-Ala-Leu97/Leu98 and Arg-Gly-Ala-Gln-Thr-Leu-Tyr186.This and other structural damage is due to peptide bondcleavages after Tyr74, Leu94, Leu97/Leu98, Tyr179, Tyr186and Phe195/Tyr196, resulting in loss of IGF binding activity.Although a corresponding < 20-kDa glycopeptide and someother expected large glycopeptides (Table 1) were not observedby ESI-MS, these nevertheless may have been present given thepoor sensitivity of ESI-MS to large heterogeneously glyco-sylated polypeptides compared with their nonglycosylatedequivalents, which has been noted previously with intactgIGFBP-6 and n-gIGFBP-6 [21].

Trypsin. Following limited digestion of gIGFBP-6 with trypsin,immunoblotting demonstrated the presence of an < 16 kDafragment (Fig. 6A) which did not bind [125I]IGF-II as assessedby Western ligand blotting (Fig. 6B). n-gIGFBP-6 was digestedto a greater extent by trypsin than gIGFBP-6 (Fig. 6A,B). Forexample, little or no intact n-gIGFBP-6 remained followingdigestion with 3 ng trypsin, whereas . 50% of gIGFBP-6remained intact (Fig. 6A,B).

ESI-MS analysis of gIGFBP-6 incompletely digested withtrypsin revealed the presence of a group of related glyco-peptides with masses of 11±12.7 kDa and three nonglyco-sylated 9 kDa fragments with masses of 9122.2 ^ 1.1 Da,9277.9 ^ 1.0 Da and 9463.0 ^ 1.0 Da. These were identifiedas related N-terminal fragments: Cys29±Lys118 (calculatedmass 9122.2 Da), Arg28±Lys118 (9278.4 Da) and Leu26±Lys118 (9462.6 Da) (Fig. 5). The glycopeptides were identifiedas glycosylated Glu119±Arg199, based on previous character-ization of IGFBP-6 glycoforms [21]. Trypsin thereforepreferentially cleaves gIGFBP-6 after Lys118 and Arg199(Fig. 5). Cleavage of gIGFBP-6 after Lys118 alone wouldproduce an < 16 kDa glycosylated Glu119±Ser239 fragmentthat would account for the major tryptic fragment observedby SDS/PAGE (Fig. 6A). Consistent with this interpretation,the 2±3 kDa lower apparent mass of the fragment generatedfrom n-gIGFBP-6 (Fig. 6A) may be due to the absence ofglycosylation.

D I S C U S S I O N

IGFBP-6 is an O-linked glycoprotein which is a relativelyspecific inhibitor of IGF-II actions [13,17,35,36]. O-glycosyla-tion delays the clearance of IGFBP-6 from the circulation [22],but it does not directly influence high affinity IGF binding byIGFBP-6 [20,23]. However, the present study demonstrates twomeans by which O-glycosylation indirectly maintains the highIGF-II binding affinity of IGFBP-6, namely by inhibitingbinding to glycosaminoglycans and by protecting againstproteolysis.

Although most studies have not detected cell-associatedIGFBP-6 [4,17], we have shown a small, inconsistent amount ofrat IGFBP-6 in cell lysates from rat PC12 cells [18]. In thepresent study, IGFBP-6 was also found in PC12 membrane

Fig. 5. Preferred chymotrypsin and trypsin cleavage sites of glyco-

sylated IGFBP-6. As shown in bold, preferred cleavage sites for partial

digestion of gIGFBP-6 by chymotrypsin (W, Y, F, L) and trypsin (K, R)

were determined by ESI-MS (Table 1). Amino acids are numbered

according to the sequence IBP6_HUMAN (accession number P24592) in

the SWISS-PROT database. O-glycosylation sites [21] are arrowed.

Cysteine disulphide linkages [30] are also indicated as solid lines.

Fig. 6. O-glycosylation protects IGFBP-6 from digestion by trypsin.

Glycosylated (G) and nonglycosylated (N) IGFBP-6 (200 ng) were digested

with trypsin for 2 h at 37 8C. Samples were analysed by SDS/PAGE

followed by (A) immunoblotting and (B) Western ligand blotting using

[125I]IGF-II on the same membranes. This figure is representative of three

independent experiments.


preparations. It may appear contradictory that rat IGFBP-6,which is also O-glycosylated [37], is membrane associated.However, rat IGFBP-6 is substantially less O-glycosylated thanhuman IGFBP-6 [37], sharing only one of the five recentlyidentified O-glycosylation sites of human IGFBP-6 [21].Furthermore, only a minor proportion of endogenously syn-thesized rat IGFBP-6 was membrane associated in PC12 cells.The inhibition of binding of both endogenous rat IGFBP-6 andexogenous human n-gIGFBP-6 to cell membranes by increas-ing salt concentrations suggests that their binding is due to ionicinteractions, which is consistent with binding to glycosamino-glycans. Further studies demonstrating decreased IGFBP-6binding to PC12 cell membranes following incubation ofthe latter with glycosidases such as chondroitinase ABC orkeratanase would strengthen this contention.

IGF-II binding after incubation of glycosaminoglycans withn-gIGFBP-6 was approximately threefold greater than withgIGFBP-6. As gIGFBP-6 and n-gIGFBP-6 have similar bindingaffinities for IGF-II in solution (present study and [20,23]) andsimilarly reduced IGF-II binding affinities when bound tochondroitin 6-sulphate (present study), this indicates thatO-glycosylation inhibits binding of IGFBP-6 to glycosamino-glycans. Similarly to IGFBP-2 [6], IGFBP-6 bound equally to arange of glycosaminoglycans. In contrast, IGFBP-3 andIGFBP-5 bind preferentially to glycosaminoglycans such asheparin, heparan sulfate, and dermatan sulfate and minimally tochondroitin sulfates and hyaluronic acid [3±5].

Glycosylation of other proteins has also been shown to beinhibit interactions with glycosaminoglycans. Site-directedmutagenesis of recombinant anti-thrombin resulting in theloss of each of four N-linked oligosaccharides increased itsheparin binding affinity two to sevenfold and it was suggestedthat glycans decrease binding affinity by direct steric effects[25]. Glycosylation of the CD44 receptor also regulates bindingto hyaluronan in a complex manner [24,38,39]. A number ofmechanisms for the effects of glycosylation were proposed,including steric effects [39], direct involvement of carbo-hydrates in the ligand binding site [24], and charge-mediatedeffects due to the presence of sialic acids [39].

IGFBP-6 shares a putative heparin binding domain withIGFBP-3 and IGFBP-5, both of which are more commonly cellassociated. A peptide based on the heparin binding domain ofIGFBP-6 competed for IGFBP-3 cell binding with similarpotency to a peptide based on this domain of IGFBP-3 but to alesser extent than a similar peptide based on IGFBP-5 [4]. It isunclear whether peptides based on the heparin binding domainof IGFBP-6 bind heparin with similar [19] or lower [40] affinitythan peptides based on IGFBP-3 and -5. Nevertheless, O-glyco-sylation may mask the heparin binding site and therebycontribute to the lack of glycosaminoglycan binding andtherefore cell association of IGFBP-6.

In the present study, glycosaminoglycan-bound IGFBP-6had a 10-fold lower binding affinity for IGF-II than solubleIGFBP-6. This is consistent with previous studies whichshowed that interaction with glycosaminoglycans decreasesthe IGF binding affinities of IGFBP-2 [6], IGFBP-3 [3,41] andIGFBP-5 [42]. It has been postulated that decreases in IGFbinding affinity are due to changes in conformation of theIGFBP following interaction with the glycosaminoglycan. Inaddition to the effects of glycosaminoglycans on IGF bindingby IGFBPs, proteolysis of IGFBPs may occur following cellassociation [31] resulting in further decreases in IGF bindingaffinity.

In this study, O-glycosylation inhibited proteolysis of humanIGFBP-6 by chymotrypsin and trypsin. This finding is

consistent with those of other studies in which glycosylationhas been shown to inhibit proteolysis, since hydrophilic,relatively bulky carbohydrates on the surface of glycoproteinsmay mask proteolytic cleavage sites [43].

The major consequence of IGFBP proteolysis is a decrease inIGF binding affinity, resulting in the release of free IGFs thatmay then bind to and activate the IGF-I receptor [7]. In thisstudy, we have shown that proteolysis of IGFBP-6 by trypsin orchymotrypsin decreases IGF-II binding, which is consistentwith the effects of proteolysis on other IGFBPs. The differentcleavage patterns of these proteases, however, suggest differentmechanisms for the loss of binding affinity. Trypsin cleavage inthe nonconserved mid-region of IGFBP-6 results in a 16-kDaC-terminal fragment which lacks the region corresponding tothe high affinity IGF binding site recently described forIGFBP-5 [44]. Previous studies have shown that C-terminalfragments of IGFBPs do not bind IGFs or bind with markedlydecreased affinity [45,46]. N-terminal fragments of IGFBPslacking the mid- and N-terminal regions, as observed followingtrypsin digestion of IGFBP-6, also have markedly reduced IGFbinding affinities [44,46,47].

In contrast, a relatively small reduction in molecular massfollowing partial digestion of IGFBP-6 by chymotrypsinresulted in dramatically decreased IGF-II binding. Althoughthe principal mass loss was due to removal of a 4.5-kDaC-terminal fragment of IGFBP-6 which may contain determi-nants of IGF binding [46,48], it is more likely that the dramaticloss of IGF binding affinity following chymotrypsin digestionof IGFBP-6 is due to cleavages involving hydrophobic aminoacids that are homologous with those in the high affinity IGFbinding site of IGFBP-5 [44].

Chymotrypsin and trypsin both belong to the family of serineproteases that includes thrombin, plasmin and kallikreins,which have been implicated in physiological cleavage ofIGFBPs [7]. For example, IGFBP-3 is cleaved in vivo byprostate-specific antigen [49], the purified form of which hasonly chymotrypsin-like activity [33]. Most cleavage sites ofIGFBP proteases are found in the nonconserved mid-regions[7], which may confer specificity of proteases for individualIGFBPs. However, cleavage of the high affinity IGF bindingsite of IGFBPs by a protease with specificity for hydrophobicamino acids could also serve functionally as an efficientmechanism for inactivating IGFBPs and releasing free IGFs. Asthis is a highly conserved region of the IGFBPs, such proteasescould possibly cleave multiple IGFBPs. Further, cleavage atthese sites may not lead to major reductions in apparentmolecular mass by nonreducing SDS/PAGE, as fragments mayremain connected by disulphide linkages. Because IGFBPproteolysis is often analysed by this method, proteolysis atthese sites may not have been detected. However, our obser-vations with chymotrypsin show that this form of IGFBPcleavage is possible. We have recently also observed a dramaticdecrease in IGF-II binding without a decrease in apparentmolecular mass of IGFBP-6 following cleavage by an acid-activated protease in keratinocytes [29]; this observation mayalso be explained by disulphide linkages holding togetherproteolytically damaged IGFBP-6.

In conclusion, the present study shows that O-glycosylationinhibits binding of IGFBP-6 to glycosaminoglycans and cellmembranes and also inhibits proteolysis of IGFBP-6 by trypsinand chymotrypsin. Both glycosaminoglycan binding and pro-teolysis of IGFBP-6 decrease its binding affinity for IGF-II.O-glycosylation therefore inhibits two potential processes bywhich the IGF-II binding affinity of IGFBP-6 may be dimin-ished. Although O-glycosylation does not affect directly the


IGF-II binding affinity of IGFBP-6 [20,23], it neverthelessappears to be involved in maintaining IGFBP-6 in a soluble,high affinity state, thereby contributing to its inhibitory func-tion. As native IGFBP-6 is found mainly, if not exclusively, inthe glycosylated form, O-glycosylation may contribute to theunique properties of this IGFBP. Further studies are required toassess the physiological importance of the findings described inthis paper.

A C K N O W L E D G E M E N T S

This work was supported by grants from the Australian Research Council

(Department of Education, Training and Youth Affairs), the Austin Hospital

Medical Research Foundation and the Sir Edward Dunlop Medical

Research Foundation. We gratefully acknowledge iodination of IGF-II by

D. Casley.

R E F E R E N C E S

1. Jones, J.I. & Clemmons, D.R. (1995) Insulin-like growth factors

and their binding proteins: biological actions. Endocr Rev. 16,

3±34.

2. Bach, L.A. & Rechler, M.M. (1995) Insulin-like growth factor binding

proteins. Diabetes Rev. 3, 38±61.

3. Arai, T., Parker, A., Busby, W. Jr & Clemmons, D.R. (1994) Heparin,

heparan sulfate, and dermatan sulfate regulate formation of the

insulin-like growth factor-I and insulin-like growth factor-binding

protein complexes. J. Biol. Chem. 269, 20388±20393.

4. Booth, B.A., Boes, M., Andress, D.L., Dake, B.L., Kiefer, M.C.,

Maack, C., Linhardt, R.J., Bar, K., Caldwell, E.E. & Weiler, J. (1995)

IGFBP-3 and IGFBP-5 association with endothelial cells: role of

C-terminal heparin binding domain. Growth Regul. 5, 1±17.

5. Fowlkes, J.L. & Serra, D.M. (1996) Characterization of glycosamino-

glycan-binding domains present in insulin-like growth factor-binding

protein-3. J. Biol. Chem. 271, 14676±14679.

6. Russo, V.C., Bach, L.A., Fosang, A.J., Baker, N.L. & Werther, G.A.

(1997) Insulin-like growth factor binding protein-2 binds to cell

surface proteoglycans in the rat brain olfactory bulb. Endocrinology.

138, 4858±4867.

7. Fowlkes, J.L. (1998) Insulinlike growth factor-binding protein pro-

teolysis. An emerging paradigm in insulinlike growth factor

physiology. Trends Endocrinol. Metab. 8, 299±306.

8. Toretsky, J.A. & Helman, L.J. (1996) Involvement of IGF-II in human

cancer. J. Endocrinol. 149, 367±372.

9. Bach, L.A. (1999) Insulin-like growth factor binding protein-6: the

`forgotten' binding protein? Horm. Metab. Res. 31, 226±234.

10. Roghani, M., Lassarre, C., Zapf, J., Povoa, G. & Binoux, M. (1991)

Two insulin-like growth factor (IGF) -binding proteins are respon-

sible for the selective affinity for IGF-II of cerebrospinal fluid

binding proteins. J. Clin. Endocrinol. Metab. 73, 658±666.

11. Forbes, B., Ballard, F.J. & Wallace, J.C. (1990) An insulin-like growth

factor-binding protein purified from medium conditioned by a human

lung fibroblast cell line (He[39]L) has a novel N-terminal sequence.

J. Endocrinol. 126, 497±506.

12. Martin, J., Willetts, K. & Baxter, R.C. (1990) Purification and

properties of a novel insulin-like growth factor-II binding protein

from transformed human fibroblasts. J. Biol. Chem. 265, 4124±4130.

13. Kiefer, M.C., Schmid, C., Waldvogel, M., Schlapfer, I., Futo, E.,

Masiarz, F.R., Green, K., Barr, P.J. & Zapf, J. (1992) Characterization

of recombinant human insulin-like growth factor binding proteins 4,

5, and 6 produced in yeast. J. Biol. Chem. 267, 12692±12699.

14. Bach, L.A., Hsieh, S., Sakano, K., Fujiwara, H., Perdue, J. & Rechler,

M. (1993) Binding of mutants of human insulin-like growth factor II

to insulin- like growth factor binding proteins 1±6. J. Biol. Chem.

268, 9246±9254.

15. Gabbitas, B. & Canalis, E. (1996) Retinoic acid stimulates the

transcription of insulin-like growth factor binding protein-6 in

skeletal cells. J. Cell Physiol. 169, 15±22.

16. Grellier, P., De Galle, B. & Babajko, S. (1998) Expression of insulin-

like growth factor-binding protein 6 complementary DNA alters

neuroblastoma cell growth. Cancer Res. 58, 1670±1676.

17. Bach, L.A., Hsieh, S., Brown, A.L. & Rechler, M.M. (1994)

Recombinant human insulin-like growth factor binding protein-6

inhibits IGF-II-induced differentiation of L6A1 myoblasts. Endo-

crinology 135, 2168±2176.

18. Bach, L.A., Leeding, K.S. & Leng, S.L. (1997) Regulation of IGF-

binding protein-6 by dexamethasone and IGFs in PC12 rat

pheochromocytoma cells. J. Endocrinol. 155, 225±232.

19. Booth, B.A., Boes, M., Dake, B.L., Linhardt, R.J., Caldwell, E.E.,

Weiler, J.M. & Bar, R.S. (1996) Structure±function relationships in

the heparin-binding C-terminal region of insulin-like growth factor

binding protein-3. Growth Regul. 6, 206±213.

20. Bach, L.A., Thotakura, N.R. & Rechler, M.M. (1992) Human insulin-

like growth factor binding protein-6 is O-glycosylated. Biochem.

Biophys. Res. Commun. 186, 301±307.

21. Neumann, G.M., Marinaro, J.A. & Bach, L.A. (1998) Identification of

O-glycosylation sites and partial characterization of carbohydrate

structure and disulfide linkages of human insulin-like growth factor

binding protein-6. Biochemistry 37, 6572±6585.

22. Marinaro, J.A., Casley, D.J. & Bach, L.A. (2000) O-glycosylation

delays the clearance of human IGF-binding protein-6 from the

circulation. Eur. J. Endocrinol. 142, 512±516.

23. Marinaro, J.A., Jamieson, G.P., Hogarth, P.M. & Bach, L.A. (1999)

Differential dissociation kinetics explain the binding preference of

insulin-like growth factor binding protein-6 for insulin-like growth

factor-II over insulin-like growth factor-I. FEBS Lett. 450, 240±244.

24. Katoh, S., Zheng, Z., Oritani, K., Shimozato, T. & Kincade, P.W.

(1995) Glycosylation of CD44 negatively regulates its recognition of

hyaluronan. J. Exp. Med. 182, 416±429.

25. Olson, S., Frances-Chmura, A., Swanson, R., Bjork, I. & Zettlmeissl,

G. (1997) Effect of individual carbohydrate chains of recombinant

antithrombin on heparin affinity and on the generation of glycoforms

differing in heparin affinity. Arch. Biochem. Biophys. 341, 212±221.

26. Varki, A. (1993) Biological roles of oligosaccharides: all of the theories

are correct. Glycobiology 3, 97±130.

27. Munson, P.J. & Rodbard, D. (1984) Computerized analysis of ligand

binding data: basic principles and recent development. Computers in

Endocrinology (Rodbard, D. & Forti, G., eds), pp. 117±145. Raven

Press. New York.

28. Bach, L.A. & Rechler, M.M. (1996) Measurement of insulin-like

growth factor (IGF) -II binding to purified IGF binding proteins 1±6:

comparison of charcoal adsorption and high performance size

exclusion chromatography. Biochim. Biophys. Acta. 1313, 79±88.

29. Marinaro, J.A., Hendrich, E.C., Leeding, K.S. & Bach, L.A. (1999)

HaCaT human keratinocytes express IGF-II, IGFBP-6 and an acid-

activated protease with activity against IGFBP-6. Am. J. Physiol. 276,

E536±E542.

30. Neumann, G.M. & Bach, L.A. (1999) The N-terminal disulfide

linkages of human insulin-like growth factor binding protein-6

(hIGFBP-6) and hIGFBP-1 are different as determined by mass

spectrometry. J. Biol. Chem. 274, 14587±14594.

31. Conover, C.A. (1991) Glycosylation of insulin-like growth factor

binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I

action: evidence for processing of cell-bound IGFBP-3. Endocrinology

129, 3259±3268.

32. Jones, J.I., Gockerman, A., Busby, W.H. Jr, Camacho Hubner, C. &

Clemmons, D.R. (1993) Extracellular matrix contains insulin-like

growth factor binding protein-5: potentiation of the effects of IGF-I.

J Cell Biol. 121, 679±687.

33. Okabe, E., Kajihara, J., Usami, Y. & Hirano, K. (1999) The cleavage

site specificity of human prostate specific antigen for insulin-like

growth factor binding protein-3. FEBS Lett. 447, 87±90.

34. Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W. & Wingfield,

P.T. (1997). Current Protocols in Protein Science. John Wiley &

Sons, New York.

35. Bach, L.A., Salemi, R. & Leeding, K.L. (1995) Roles of insulin-

like growth factor (IGF) receptors and IGF binding proteins in


IGF-II-induced proliferation and differentiation of L6A1 rat

myoblasts. Endocrinology 136, 5061±5069.

36. Schmid, C., Schlapfer, I., Keller, A., Waldvogel, M., Froesch, E.R. &

Zapf, J. (1995) Effects of insulin-like growth factor (IGF) binding

proteins (BPs)-3 and -6 on DNA synthesis of rat osteoblasts: further

evidence for a role of auto-/paracrine IGF I but not IGF II in

stimulating osteoblast growth. Biochem. Biophys. Res. Commun. 212,

242±248.

37. Bach, L.A., Tseng, L., Swartz, J.E. & Rechler, M.M. (1993) Rat PC12

pheochromocytoma cells synthesize insulin-like growth factor-

binding protein-6. Endocrinology 133, 990±995.

38. Skelton, T.P., Zeng, C., Nocks, A. & Stamenkovic, I. (1998)

Glycosylation provides both stimulatory and inhibitory effects on

cell surface and soluble CD44 binding to hyaluronan. J. Cell Biol.

140, 431±446.

39. English, N.M., Lesley, J.F. & Hyman, R. (1998) Site-specific de-N-

glycosylation of CD44 can activate hyaluronan binding, and CD44

activation states show distinct threshold densities for hyaluronan

binding. Cancer Res. 58, 3736±3742.

40. Fowlkes, J.L., Thrailkill, K.M., George Nascimento, C., Rosenberg,

C.K. & Serra, D.M. (1997) Heparin-binding, highly basic regions

within the thyroglobulin type-1 repeat of insulin-like growth factor

(IGF) -binding proteins (IGFBPs)-3 -5, and -6 inhibit IGFBP-4

degradation. Endocrinology 138, 2280±2285.

41. Koedam, J.A., Hoogerbrugge, C.M., van Buul Offers, S.C. & Van den

Brande, J.L. (1997) Solid-phase assay for insulin-like growth factor

(IGF) binding to IGF-binding protein-3: application to the study of

the effects of antibodies and heparin. J. Endocrinol. 153, 87±97.

42. Arai, T., Clarke, J., Parker, A., Busby, W. Jr, Nam, T. & Clemmons,

D.R. (1996) Substitution of specific amino acids in insulin-like

growth factor (IGF) binding protein 5 alters heparin binding and its

change in affinity for IGF-I response to heparin. J. Biol. Chem. 271,

6099±6106.

43. Bernard, B.A., Newton, S.A. & Olden, K. (1983) Effect of size and

location of the oligosaccharide chain on protease degradation of

bovine pancreatic ribonuclease. J. Biol. Chem. 258, 12198±12202.

44. Kalus, W., Zweckstetter, M., Renner, C., Sanchez, Y., Georgescu, J.,

Grol, M., Demuth, D., Schumacher, R., Dony, C., Lang, K. & Holak,

T.A. (1998) Structure of the IGF-binding domain of the insulin-like

growth factor- binding protein-5 (IGFBP-5): implications for IGF and

IGF±I receptor interactions. EMBO J. 17, 6558±6572.

45. Lalou, C., Lassarre, C. & Binoux, M. (1996) A proteolytic fragment of

insulin-like growth factor (IGF) binding protein-3 that fails to bind

IGFs inhibits the mitogenic effects of IGF-I and insulin. Endocrinology

137, 3206±3212.

46. Qin, X., Strong, D.D., Baylink, D.J. & Mohan, S. (1998) Structure±

function analysis of the human insulin-like growth factor binding

protein-4. J. Biol. Chem. 273, 23509±23516.

47. Hashimoto, R., Ono, M., Fujiwara, H., Higashihashi, N., Yoshida, M.,

Enjoh-Kimura, T. & Sakano, K. (1997) Binding sites and binding

properties of binary and ternary complexes of insulin-like growth

factor-II (IGF-II), IGF-binding protein-3, and acid-labile subunit.

J. Biol. Chem. 272, 27936±27942.

48. Forbes, B.E., Turner, D., Hodge, S.J., McNeil, K.A., Forsberg, G. &

Wallace, J.C. (1998) Localization of an insulin-like growth factor

(IGF) binding site of bovine IGF binding protein-2 using disulfide

mapping and deletion mutation analysis of the C-terminal domain.

J. Biol. Chem. 273, 4647±4652.

49. Cohen, P., Graves, H.C.B., Peehl, D.M., Kamerei, M., Giudice, L.C. &

Rosenfeld, R.G. (1992) Prostate specific antigen is an IGF binding

protein-3 (IGFBP-3) protease found in seminal plasma. J. Clin.

Endocrinol. Metab. 74, 743±750.


o-glycosylation of insulin-like growth factor (igf) binding protein-6 maintains high igf-ii binding...

Documents