dodson 1998

189

The role of assembly in insulin's biosynthesis Guy Dodson*t and Don Steiner

Insulin is synthesised as a single-chain precursor, preproinsulin, that contains an N-terminal signal sequence and a connecting peptide linking the A and B chains of the insulin molecule. Nascent proinsulin is directed into the regulated secretory pathway, converted to insulin and stored as microcrystals. These processes exploit assembly to the zinc- containing hexamer. Structural, chemical and genetic studies, and experiments with transgenic animals and transfected cells, are providing new details about the molecular events in insulin's biosynthesis.

Addresses *National Institute of Medical Research, Mill Hill, London, NW7 1AA, UK; e-mail: [email protected] *Chemistry Department, University of York, York YO1 5DD, UK e-mail: [email protected] ~+The Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA

Current Opinion in Structural Biology 1998, 8:189-194

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© Current Biology Ltd ISSN 0959-440X

In t roduct ion Since the discovery of insulin in 1921 by Banting and Best, a wealth of chemical, biochemical and structural knowl- edge about the hormone and its biosynthetic precursor, proinsulin, has become available. Thus, its biological and physiological effects have been characterised, its solution properties described in detail, its amino acid sequence and three-dimensional structure determined, and its chemical and recombinant synthesis established. Analyses of biological and structural research have identified the residues involved in the molecule 's self-assembly and those involved in its binding to its receptor. (For reviews of insulin's biology, chemistry and structure see [1-3].) It has been possible to take the implications of the hormone's structure further. Electron microscopic studies, time- course experiments and chemical reactivity, as well as the important development of research with transgenic mice and transfected cells, together with an understanding of insulin and proinsulin assembly, have allowed us to deduce in detail some of the fundamental chemical and physical events that occur during the hormone's biosynthesis, and their locations in the cell. Thus, we can now describe many aspects of insulin's biosynthesis at both the molecular level, and the cellular level.

In this review, we shall relate the structure and assembly of insulin to the major events of its biosynthesis. These begin with preproinsulin, its processing to proinsulin, which is then folded and subsequently assembles to a hexamer, facilitating transport. Hexameric proinsulin is converted to insulin, which then forms microcrystals in the

storage residues; uhimately it is released from the 13 cell into the blood.

Insul in's st ructure and a s s e m b l y Although insulin circulates in the serum and binds to its receptor as a monomer, it forms dimers at micromolar con- centrations, and in the presence of zinc ions it further assembles to hexamers [1]. T h e hormone's solution and crystal structures and its pattern of assembly are illustrated in Figure 1. T h e insulin monomer itself consists of two chains, an A chain of 21 amino acids and a B chain of 30 amino acids. T h e B chain has a central helical segment between residues B9 and B19, from which the N and C termini extend as strands. T h e A chain has an N-terminal helix, A1-A8, linked to an antiparallel C-terminal helix, A12-A20. T h e two chains are joined by two disulphide bonds, AT-B7 and A20-B19, which clamp the A chain helices at each end of the central B chain helix. Note that the A chain N terminus and the B chain C terminus are both located on the molecule's surface, about 8 A apart. Figure 2 shows the A chain N terminus and B chain C terminus, which in proinsulin are linked by a connecting peptide. T h e structures of the termini in the monomer (Figure 2a) and the hexamer (Figure 2b) are essentially the same. The solution structure of insulin monomers (Figure 2c) is also essentially identical to those of insulin monomers within the dimer and hexamer as determined by X-ray analysis [4"].

As is indicated in Figure 2, the insulin monomer has two extensive nonpolar surfaces. One surface is flat, well defined and mainly aromatic; it is buried upon dimer formation. T h e other is more extensive and complex and it has some flexibility. This extensive surface is buried when the dimers further assemble to hexamers in the presence of zinc ions [1]. Proinsulin and insulin have essentially the same association behaviour; thus, any significant stable contacts made by the connecting peptide must be limited to the surface and not buried in the hexamer (see Figure 3; [5,6]). T h e closeness of the two junctions also requires that the connecting polypeptide chain linking them takes a complex course on the insulin hexamer surface. Spectroscopic and N M R studies indicate that there is no convincing evidence for secondary structure in the connecting polypeptidc, and neither X-ray analysis nor N M R studies have been successful in defining its probably mobile structure. We shall see, however, that proinsulin's self assembly is beautifully exploited in the different stages of biosynthesis.

Insul in's b iosynthes is in the cell T h e biosynthesis of insulin has been studied for many years, and a good deal has been written on the various celhdar, physiological and structural aspects [2,3,5,6].

190 Macromolecular assemblages

Figure 1

B13 BIO

BIO B13

.J

i ~ L Zn2+

Monomers Dimer Hexamer

Current Opinion in Structural Biology

The pattern of assembly of insulin monomers, dimers and hexamers. Only the backbone and selected sidechains important in assembly are shown. The B10 histidine that coordinates the two central zinc ions is indicated; this residue always associates with zinc in the hexamers [5]. Six B13 glutamate residues are buried in the hexamer within hydrogen bonding distance of each other. Their unfavourable interactions are important in hexamers released into the blood.

Insulin is synthesised in the 18 cells of the islets of Langerhans. The 13 cells are characterised by two features associated with cells that export proteins - - they have rough endoplasmic reticular surfaces and well-defined storage vesicles. In this case, the vesicles typically contain microcrystals of packaged insulin. 18 cells have another important feature - - in almost all animals they are rich in zinc and calcium. Biochemical experiments and radioisotopic chase studies have determined the location and duration of the different stages of the process from the initial event of polypeptide synthesis to the appearance of crystalline insulin in a granule within the vesicle (Figure 3). These studies also show the corresponding molecular events that occur during biosynthesis [5]. The final exocytotic release of insulin from the 13-cell storage vesicles into the blood is an energy-dependent process governed by the sugar level in the blood.

The ribosomal synthesis of proinsulin The synthesis of the insulin molecule depends on the normal mechanisms for exported proteins [2]. Its mes- senger RNA contains sequences that correspond to a leader or signal sequence that is responsible for stabilis- ing the ribosome on the rough endoplasmic reticulum (RER) membrane. This is followed by a sequence that translates into the B chain and then a sequence that translates into the 30-35 amino acid connecting peptide segment, which links to the following A-chain sequence. Thus insulin is directed into the endoplasmic volume as a single-chain molecule of some 110 amino acids, referred to as preproinsulin. The signal sequence is

thought to be immediately removed to generate proinsulin. In the special environment of the ER, proinsulin is able to fold quickly into its correct three-dimensional structure, or it may fold while anchored to the membrane, as this could help localise the nascent molecules conveniently for assembly, for example as discussed in [7] with respect to haemagglutinin folding.

The nature of the environment in the ER is only begin- ning to be understood in detail. There is evidence (from the reactivity of proinsulin to iodination and disulphide reduction) that proinsulin assembly to dimers and hexamers could occur here, though questions as to the availabil- ity of zinc are unsettled [8,9]. The iodination studies exploited the altered folding properties of mutant SerB5 proinsulin that allow it to be iodinated in the ER [9]. Incidentally, this behaviour suggests that BS, normally a histidine residue, is important in folding and hexamer formation. It may further imply that the hexamer uses HisB5 in zinc binding in vivo as has been observcd in the insulin crystal that contains four zinc ions. Possibly this extra zinc interaction will further stabilise the assembly in the vesicle [10].

The transport of proinsulin into the Golgi The vesicular transfer of proinsulin from the ER brings it to the Golgi, an aqueous environment containing zinc and calcium [11]. Thus, from here on, the solution conditions will favour formation of the zinc-containing proinsulin hexamer [8]. The surface structure of the proinsulin hexamer is quite different to that of insulin as the schematic in

Figure 2

(a)

A1-3 B30

I

(b) B30

I

Nonpolar surfaces (crystal)

Monomer extracted from hexamer

The role of assembly in insulin's biosynthesis Dodson and Steiner 191

A1-3 (c)

B30

I A1-3

'iS-.

f Nonpolar surfaces Nonpolar surfaces

(crystal) (solution)

Hexamer Monomer


The insulin monomer and hexamer structures [1]. The N terminus of the A chain (A1-3) and the B chain (B30) are drawn with van der Waals' radii. These are the sites of the connecting peptide junctions in proinsulin. (a) The crystal monomer isolated from the hexamer. (b) The two-zinc insulin hexamer with the selected monomer highlighted. (c) The solution structure of the insulin monomer. On each side of the molecule are the two nonpolar surfaces that are buried by assembly to the dimer and hexamer [3]. The two-zinc hexamer is viewed perpendicular to its threefold axis; the monomers are viewed in the same direction.

Figure 3 shows, although they do share the same organisation and internal structure. This explains their profoundly different solubilities in the presence of zinc in which the proinsulin hexamer is soluble and the insulin hexamer is insoluble [11,12]. Probably, the extra four to six carboxy- late groups in each proinsulin monomer are arranged to form metal ion binding sites [5]. This solubility favours the transport of concentrated proinsulin (=10%)into the Golgi apparatus in which it is sequestered into evaginations that develop and bud off" into storage vesicles [5,6].

Expcriments on the sccretion of dimeric mutant proinsulins in transgenic animals suggest that for efficient sorting proinsulin must have a low affinity for the insulin receptor. This affinity will be provided by the proinsulin connecting peptide and additionally by the hexamer structure [13,14]. Similar studies on monomeric proinsulin using AtT-20 transfected pituitary cells have provided fllrther evidence for thc sorting of the dimeric AspB10 proinsulin into the constitutive secretory pathway [15]. Experiments with these cells using a monomeric insulin mutant (SerB9 --+ Asp) indicate that there is normal sorting [161. An explanation for this may be that AspB9 insulin has only 30% of its receptor binding

ability, which will rednce the receptor's capacity to divert the mutant proinsulin from the regulated secretory pathway. It also follows that normal sorting can be efficient without presence of the hexamer. In the presence of zinc, calcium and various additives, however, this mutant can form hexamers and this possibility should be considered [17]. More flmdamentally these transfected pituitary cells do not express high levels of insulin and are not enriched with zinc, which presumably means that there may not be hexamers. Their absence will probably affect the secretory events in the Golgi and perturb the normal concentration, precipitation and crystallisation events that occur in the Golgi and storage vesicles of the ~ cell.

In another experiment, a single chain insulin in which A1 was directly linked to B30 was used. This proinsulin ana- logue is closely related to the A1-B29 cross-linked insulin that forms hexamers and crystallises readily [18-20]. This molecule, essentially a truncated proinsulin from which the connecting peptide segment has been deleted, has very low binding affinity for the receptor and thus will sort efficiently. It would be interesting to measure the efficiency of this truncated proinsulin's biosynthesis and


Figure 3

Molecular events Cellular events

Reduced unfolded preproinsulin

SS bond formation, proinsulin folding

Formation of zinc proinsulin hexamers

zinc insulin hexamer with released C peptides Precipitation begins

-k Crystal formation

l I ~ W _~ f Amino acids RER •\ "~ J Transfer q/ I .... / RNA ATP, GTP, Mg ++

"~ Enzymes

Proinsulin (SS bond i formation) ~ t ...... Antimycin blocks

© O .... MV % O O ~1~"" Transfer step1 (energy dependent)

O o o Proinsulin O O O

O O O

o ° ° o -- Transfer step 2

Membrane "~ bound }, .... proteases 3

Zinc++'~-----~ ~I I

Mainly insulin ~ ~ | (crystalloid) . \ / I |

+ ~ . ~ ~ ¢ I 1 0 ~. Membrane C e tide ~ p P' ~ ' ~ . ~ @ ~' f-"~ | "~ recycling?

• - - ature , , . (space) ~ granules

PmlaSmb:a n e ~

~ |

E m i o c y t o s i ~ Y e d p ~ P n ~ n e d n l ) nt

10-20 min

20 min

30-120 min

Hours-days I

(Exocytosis) Secreted products

Insulin } 94% C peptide Proinsulin 'l t _60/0 intermediates " Zn ++ Others? Current Opinion in Structural Biology

A schematic representation of the molecular events (left) and cellular events (right) that occur in insulin's biosynthesis. The asterisk indicates the bound or (C peptide) free connecting peptide. RER, rough endoplasmic reticulum.

transport through the ER and Golgi compared to that of normal proinsulin.

Experiments on sulfhydryl accessibility of proinsulin in pancreatic 13 cells add further direct evidence about zinc

localisation and its effects on proinsnlin assembly. Huang and Arvan [8] have shown that zinc protects proinsulin from reduction in the Golgi but not in the ER, consistent with the idea that proinsulin hexamers are stabilised both by zinc coordination to the B10 histidines and by zinc

The role of assembly in insulin's biosynthesis Dodson and Steiner 193

binding at other sites [5,12]. These observations are in con- flict with those derived from the iodination experiments of Schmitz et a/. [9].

Storage granule formation It is apparent from radiolabelling and electron microscopic studies that the conversion of the proinsulin to the insulin starts at the stage of vesicle formation [2,3]. T he connecting peptide that links the A and B chains then floats flee as the so called C peptide (see Figure 3), T he cleavage is car- ried out by membrane-associated enzymes [21]. The presence of zinc ions and the stability of the hexamer makes it likely that this hcxameric organisation will be retained throughout the conversion process. Figure 2 shows that the sites of cleavage at the A chain N terminus and B chain C terminus jtmctions are about as exposed in the hexamer as in the monomer, though they will be less flexible. Support for conversion of the hcxamer is given by the ability of trypsin to convert dimeric and hexameric proinsulin at the same rate [,5]. Electron micmgraphs sometimes show pre- cipitating material at the perimeter of the vesicle, next to the membrane [22]. This material is presumably newly converted hexameric insulin, which is insoluble in the presence of zinc ions. This is in total contrast to the pronounced solubility of proinsulin hexamers [12].

There is a further advantage with retaining the hexamcr: the six insulin molectdes form an oblate spheroid that readily fl)rms close-packed arrays that favour crystal growth. Thus on precipitation in the vesicle, insulin hexamers tend to form microcrystals, behaviour that has been duplicated in laboratory experiments [23-2,5]. Storage vesicles of insulins that are dimeric or monomeric, that is, those of the hagfish or guinea pig, respectivcb; do not contain crystals but rather electron dense amorphous precipitates [,5,6,22]. Equally, in transgenic mice that express dimeric insulins and native hcxameric insulins, the storage

Figure 4


Electron micrographs of ~ cells of (a) normal and (b) HisB10Asp proinsulin transgenic mice. Pancreas pieces were fixed in Kanovsky's paraformaldehyde-glutaraldehyde, postfixed in 1% osmium tetraoxide and embedded in Epon. Bars represent one micron.

vesicles often have amorphous contents although somc- timcs therc are poorly formed crystals, as is illustrated in Figure 4. This shows that crystallisation has been compro- miscd by the presence of mutated insulins [13].

The appearance of crystals in a biological process is unex- pected and it is reasonable to expect that they have a function. One such function may well be to protect insulin from fllrther proteolysis by converting enzymes. Another is the selection of fnlly converted insulin hexamers by the crystalline lattice - - this would help drive the conversion of all six subunits to about 95% completion. The crystal lattice, however, does not seem to be important for conversion efficiency. In experiments on transgenic mice, it has been fi)und that hexameric and dimeric insulins are converted at much the same rate and efficient3, exen though most of the vesicles apparently do not contain crystalline material but rather amorphous precipitates [13]. It may be relevant that the dimeric AspB10 mutant insulin crystallises in the presence of zinc ions to form a dodecamer in which six dimers are arranged around two central zinc ions coordi- nated in a novel fashion to the B5 histidines [17]. There is no evidence from electron micmgraphs [13], however, that this kind of crystal is present in the storage vesicles although it is possible that their assembly in solution could affect or even fawmr conversion.

The release of the active monomeric insulin from the hexamer Within the storage vesicle, the insulin hexamer in the crystalline state is very stable [26]. On release into the serum, however, the insulin microcrystals experience a jump in pH from = 5.,5 to 7.4. As can be seen in Figure 1, the car- boxylic acids of the six GIuB13 residues are packed closely together at the centre of the hexamer. At a pH of = 6 they are hydrogen bonded to each other [11; at the pH of blood, 7.4, these interactions are increasingly disfavoured by the deprotonation of the carbnxylic acids and the zinc coordination is essential to contain their repulsion. At the same time, the dilution of the zinc and calcium ions in the serum means that the central zinc ions, which exchange on a time scale of seconds, disappear. The strong mutual repulsion of the six central B13 carboxylates canses the hexamers and therefore the crystal to disintegrate rapidly.

The rnle of B13 in destabilising the hexamer can be demonstrated by the consequences of its mutation to glut- amine, which is neutral and can be accommodated in the hexamer core. The hexamer forms in the absence of zinc and has been crystallised and its structure determined [191. This hexamer, and the crystal, would presumably not disintegrate on release from a storage vesicle and hence would delay the appearance of the monomer.

C o n c l u s i o n s In all but a few species, ~ cells contain high levels of zinc and correspondingly their insulins arc able to form zinc- containing hexamers. There is a succession of steps in


insulin's biosynthesis that appear likely to depend on the structural organisation of the pmhormone and hormone hexamer. The combined evidence provided by the hexamer structure, its chemical behaviour and its solution properties makes a persuasive case for a role for the hexamer in many of the biosynthetic processes of the 13 cell. The evidence from site-directed proinsulin and insulin mutants in transgenic animals and transfection experiments have added greatly to the arguments concerning the role of the hexamer in the Golgi and storage vesicles. There are, however, still outstanding questions: the nature of the aggrega- tion in the ER, the precise determinants in sorting, the role of the connecting peptide in transport and conversion, and the role of insulin crystallisation in the conversion process, are just some examples.

The mutations in proinsulin and insulin that modify hexamer solubility and stability and those that alter the length and nature of the connecting peptide need more investiga- tion. These studies should extend to transgenic animals and ideally should include transfected 13 cells in which the normal proinsulin gene has been deleted, allowing uncom- plicated analysis of the effects of the proinsulin mutations on biosynthesis. The use of chemical reagents that block the various biosynthetic steps and the exploitation of spe- cific reactions with the proinsulin molecule are starting to provide a more precise description of its assembly within the biosx/nthetic process. There are obvious difficulties with the chemical approach; the different patterns of reac- tion with disulphide reduction and iodination are evidence of this. Nonetheless, this approach promises to penetrate the complexity of the chemical, structural and celhdar fac- tors that govern insulin's biosynthesis.

Acknowledgements The authors are grateful to Stephen Mumfurd for preparing the figurcs, and to Hewson Swift, who made the electron micro~,raphs.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest *" of outstanding interest

1. Baker EN, Blundell TL, Cutfield JF, Cutfield SM, Dodson EJ, Dodson GG, Hodgkin DC, Hubbard RE, lsaacs NW, Reynolds CD etaL: The structure of 2 Zn pig insulin crystals at 1.5A. Philos Trans R Soc Lond Biol 1988, 319:369-456.

2. Steiner DF: The biosynthesis of insulin: genetic, evolutionary and pathophysiologic aspects, In The Harvey Lectures. New York: Academic Press; 1981,78:191-228.

3. Steiner DF, Bell GI, "lager HS, Rubenstein AH: Chemistry and biosynthesis of the islet hormones. In Endocrinology. Edited by De Groot L. London: WB Saunders; 1995:1296-1328.

4. Olsen HB, Ludvigsen S, Kaarsholm NC: Solution structure of an engineered insulin monomer at neutral pH. Biochemistry 1996, 35:8836-8845.

The solution structure derived from NMR spectroscopy of a mutant insulin monomer at neutral pH is described. There is great similarity between this well-defined structure and the molecules in the dimers and T6 hexamers determined by X-ray analysis. In particular the A and B-chain termini are seen to be little changed in the monomer, consistent with the thinking that conversion can occur in the hexamer.

5. Emdin SF, Dodson GG, Cutfield JF, Cutfield SM: Role of zinc insulin biosynthesis. Diabetologia 1980, 19:174-182.

6. Blundell TL, Dodson GG, Hodgkin DH, Mercola DA: Insulin: the reflection in the crystal and its reflection in chemistry and biology. Adv Protein Chem 19?2, 26:280-422.

7. Wilson IA, Skehel J J, Wiley DC: Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolution. 1981, 289:366-373.

8. Huang XF, Arvan P: Intrecellular transport of proinsulin in pancreatic B-cells. J Biol Chem 1995, 270:2041 ?-20423.

9. Schmitz A, Maintz M, Kehle T, Herzog V: In vivo iodination of a misfolded proinsulin reveals co-localised signals for Bip binding and for degradation in the ER. EMBO J 1995, 14:1091-1098.

10. Smith GD, Swenson DC, Dodson E J, Dodson GG, Reynolds CD: Structural stability in the 4Zn human insulin hexamer. Proc Nat/ Acad Sci USA 1994, 81:7093-709?.

11. Howell SL, Tyhurst M, Duvefelt H, Andersson A, Hellerstrom C: Role of zinc and calcium in the formation and storage of insulin in the pancreatic B-cell. Cell Tissue Res 19?8, 188:107-118.

12. Grant PT, Coombs TL, Frank BH: Differences in the nature of the interaction of insulin and proinsulin with zinc. Biochem J 1971, 126:433-440.

13. Carroll R J, Hammer RE, Chan S J, Swift HH, Rubenstein AH, Steiner DF: A mutant proinsulin is secreted from the islets of Langerhans in increased amounts via an unregulated pathway. Proc Nat/Acad Sci USA 1988, 85:8943-8947.

14. Steiner DE Tager HS, Nanjo K, Chan S J, Rubenstein AH: Familial syndromes of hyperproinsulinemia and hyperinsulinemia with mild diabetes. In The Metabofic and Molecular Bases of Inherited Disease, vol 1. Edited by Scriver CR, Baudet AL, Sly WS, Stanbury JB, Wyngaarden JB, Fredrickson. New York: McGraw Hill; 1995:897-904.

15. Gross D J, Halban PA, Kahn RC, Weir GC, Villa-Komaroff L: Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT- 20 cells. Proc Nail Acad Sci USA 1989, 96:4107-4111.

16. Quinn D, Orci L, Ravazzola M, Moore HPH: Intracellular transport and sorting of mutant proinsulins that fail to form hexamers. J Cell Bio11991,113:987-996.

17. Dodson E J, Dodson GG, Hubbard RE, Moody PCE, Turkenburg JT, Whittingham JL, Xiao B, Brange J, Kaarsholm N, Thogerson H: Insulin assembly: its modification by protein engineering and ligand binding. Philos Trans R Soc Lond A Phys Sci Eng 1993,345:153-164

18. Powell SK, Orci L, Craik CS, Moore MHM: Efficient targeting to storage granules of human proinsulins with altered propeptide domains. J Cell Biol 1988, 106:1843-1851

19. Bentley GA, Brange J, Dodson E J, Dodson GG, Wilkinson A J, Xiao B: The role of B13 glutamic acid in assembly. J Mol Biol 1992, 228:1163-1176.

20. Derewenda U, Derewenda Z, Dodson E J, Dodson GG, Bing X, Markussen J: The X-ray analysis of the single chain A1-B29 peptide linked insulin molecule. J Mol Biol 1991,220:425-433.

21. Bennet DL, Bailyes EM, Nielsen E, Guest P, Rutherford NG, Arden SD, Hutton JC: Identification of the type-2 proinsulin processing endopeptidase as PC2, a member of the eukaryotic subtilisin family../Bie/Chem 1992, 267:15229-15236

22. Sato T, Herman L, Fitzgerald PJ: The comparative ultrastructure of the pancreatic islet of Langerhans. Gen Comp Endocrino/1966, 7:132-157.

23. Schlichtkrull J: Insulin crystals [PhD Thesis]. Munskgaard: University of Copenhagen; 1958.

24. Brange J: Galenics of Insulin. Heidelberg: Springer-Verlag; 1987.

25. Michael J, Carroll R, Swift HH, Steiner DF: Studies on the molecular organization of rat insulin secretory granules. J Biol Chem 1987, 262:16531-16535.

26. Coore HG, Hellman B, Taljedal IB: Preparation and properties of isolated mammalian insulin storage granules. In The Structure and Metabolism of the Pancreatic Islets. Edited by Falkmer S, Hellman B, Taljedal B. Oxford: Pergamon Press; 1970.

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