Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

Structural heterogeneity of type I collagen triple helix and its role in osteogenesis imperfecta

We investigated regions of different helical stability within human type I collagen and discussed their role in intermolecular interactions and osteogenesis imperfecta (OI). By differential scanning calorimetry and circular dichroism, we measured and mapped changes in the collagen melting temperature (DeltaTm) for 41 different Gly substitutions from 47 OI patients. In contrast to peptides, we found no correlations of DeltaTm with the identity of the substituting residue. Instead, we observed regular variations in DeltaTm with the substitution location in different triple helix regions. To relate the DeltaTm map to peptide-based stability predictions, we extracted the activation energy of local helix unfolding (DeltaG) from the reported peptide data. We constructed the DeltaG map and tested it by measuring the H-D exchange rate for glycine NH residues involved in interchain hydrogen bonds. Based on the DeltaTm and DeltaG maps, we delineated regional variations in the collagen triple helix stability. Two large, flexible regions deduced from the DeltaTm map aligned with the regions important for collagen fibril assembly and ligand binding. One of these regions also aligned with a lethal region for Gly substitutions in the alpha1(I) chain.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

Template‐tethered collagen mimetic peptides for studying heterotrimeric triple‐helical interactions

Collagen mimetic peptides (CMPs) have been used to elucidate the structure and stability of the triple helical conformation of collagen molecules. Although CMP homotrimers have been widely studied, very little work has been reported regarding CMP heterotrimers because of synthetic difficulties. Here, we present the synthesis and characterization of homotrimers and ABB type heterotrimers comprising natural and synthetic CMP sequences that are covalently tethered to a template, a tris(2‐aminoethyl) amine (TREN) succinic acid derivative. Various tethered heterotrimers comprising synthetic CMPs [(ProHypGly)₆, (ProProGly)₆] and CMPs representing specific domains of type I collagen were synthesized and characterized in terms of triple helical structure, thermal melting behavior, and refolding kinetics. The results indicated that CMPs derived from natural type I collagen sequence can form stable heterotrimeric helical complexes with artificial CMPs and that the thermal stability and the folding rate increase with the increasing number of helical stabilizing amino acids (e.g. Hyp) in the peptide chains. Covalent tethering enhanced the thermal stability and refolding kinetics of all CMPs; however, their relative values were not affected suggesting that the tethered system can be used for comparative study of heterotrimeric CMP's folding behavior in regards to chain composition and for characterization of thermally unstable CMPs. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 94–104, 2011.     

Collagen model peptides: Sequence dependence of triple-helix stability

The triple helix is a specialized protein motif, found in all collagens as well as in noncollagenous proteins involved in host defense. Peptides will adopt a triple-helical conformation if the sequence contains its characteristic features of Gly as every third residue and a high content of Pro and Hyp residues. Such model peptides have proved amenable to structural studies by x-ray crystallography and NMR spectroscopy, suitable for thermodynamic and kinetic analysis, and a valuable tool in characterizing the binding activities of the collagen triple helix. A systematic approach to understanding the amino acid sequence dependence of the collagen triple helix has been initiated, based on a set of host-guest peptides of the form, (Gly-Pro-Hyp)(3)-Gly-X-Y-(Gly-Pro-Hyp)(4). Comparison of their thermal stabilities has led to a propensity scale for the X and Y positions, and the additivity of contributions of individual residues is now under investigation. The local and global stability of the collagen triple helix is normally modulated by the residues in the X and Y positions, with every third position occupied by Gly in fibril-forming collagens. However, in collagen diseases, such as osteogenesis imperfecta, a single Gly may be substituted by another residue. Host-guest studies where the Gly is replaced by various amino acids suggest that the identity of the residue in the Gly position affects the degree of destabilization and the clinical severity of the disease.     

Dissecting a bacterial collagen domain from Streptococcus pyogenes: sequence and length-dependent variations in triple helix stability and folding

To better investigate the relationship between sequence, stability, and folding, the Streptococcus pyogenes collagenous domain CL (Gly-Xaa-Yaa)(79) was divided to create three recombinant triple helix subdomains A, B, and C of almost equal size with distinctive amino acid features: an A domain high in polar residues, a B domain containing the highest concentration of Pro residues, and a very highly charged C domain. Each segment was expressed as a monomer, a linear dimer, and a linear trimer fused with the trimerization domain (V domain) in Escherichia coli. All recombinant proteins studied formed stable triple helical structures, but the stability varied depending on the amino acid sequence in the A, B, and C segments and increased as the triple helix got longer. V-AAA was found to melt at a much lower temperature (31.0 °C) than V-ABC (V-CL), whereas V-BBB melted at almost the same temperature (～36-37 °C). When heat-denatured, the V domain enhanced refolding for all of the constructs; however, the folding rate was affected by their amino acid sequences and became reduced for longer constructs. The folding rates of all the other constructs were lower than that of the natural V-ABC protein. Amino acid substitution mutations at all Pro residues in the C fragment dramatically decreased stability but increased the folding rate. These results indicate that the thermostability of the bacterial collagen is dominated by the most stable domain in the same manner as found with eukaryotic collagens.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Crystal structure of the collagen model peptide (Pro‐Pro‐Gly)4‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)4 at 1.0 Å resolution

The single‐crystal structure of the collagen‐like peptide (Pro‐Pro‐Gly)₄‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)₄, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple‐helical structure and that its conformation is very similar to that of (Gly‐Pro‐Hyp)₉, which has the typical repeating sequence in collagen. High‐resolution studies on other collagen‐like peptides have shown that imino acid‐rich sequences preferentially adopt a 7/2 triple‐helical structure (θ = 51.4°), whereas imino acid‐lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly‐Hyp‐Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro‐Pro‐Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2‐helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N‐terminus of an adjacent molecule, causing axial displacement, reminiscent of the D‐staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436–447, 2013.     

Triple-helical peptides: an approach to collagen conformation, stability, and self-association

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.     

Sequence specific thermal stability of the collagen triple helix

Theoretical calculations of the thermal stability of collagen triple helices using empirical values for the contribution of individual tripeptide units are presented and compared with direct measurements of the thermal stability of various types of collagens. Relative stabilities are assigned to the positions of the tripeptide units in the amino acid sequence along the length of the collagen molecule. The sequence specific relative stabilities of type I and type XI collagens are compared. These offer insight into the reasons for the existence of unfolding intermediates in type XI collagen that are absent in type I collagen. The pattern of relative stabilities calculated for mouse type IV collagen is consistent with experimental results which indicate that the amino terminal region is very stable and that the interruptions cause increased flexibility and independently unfolding domains. Mutations in the triple helical domain of human type I procollagen occurring in brittle bone disease (osteogenesis imperfecta) show varying effects on the thermal stability of the molecule. The sequence specific thermal stability calculations shed some light on why some mutations of cysteine for glycine have greater effects on the thermal stability than others.     

Automated design of the surface positions of protein helices.

Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).     

Sequence analysis of a full-length cDNA for the murine pro alpha 2(I) collagen chain: comparison of the derived primary structure with human pro alpha 2(I) collagen.

Comparison of the nucleotide sequence and primary structure of murine and human pro alpha 2(I) collagen indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-pro-peptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]338, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human pro alpha 2(I) collagen triple helices, with no truly nonconservative substitutions.     

Folding delay and structural perturbations caused by type IV collagen natural interruptions and nearby Gly missense mutations

The standard collagen triple helix requires Gly as every third residue in the amino acid sequence, yet all nonfibrillar collagens contain sites where this repeating pattern is interrupted. To explore the effects of such natural interruptions on the triple helix, a 4- or 15-residue sequence from human basement membrane type IV collagen was introduced between (Gly-Xaa-Yaa)(n) domains within a recombinant bacterial collagen. The interruptions had little effect on melting temperature, consistent with the high thermal stability reported for nonfibrillar collagens. Although the 4-residue interruption cannot be accommodated within a standard triple helix, trypsin and thermolysin resistance indicated a tightly packed structure. Central residues of the 15-residue interruption were protease-susceptible, whereas residues near the (Gly-Xaa-Yaa)(n) boundary were resistant, supporting a transition from an alternate conformation to a well packed triple helix. Both interruptions led to a delay in triple-helix folding, with the 15-residue interruption causing slower folding than the 4-residue interruption. These results suggest that propagation through interruptions represents a slow folding step. To clarify the relation between natural interruptions and pathological mutations, a Gly to Ser missense mutation was placed three triplets away from the 4-residue interruption. As a result of this mutation, the 4-residue interruption and nearby triple helix became susceptible to protease digestion, and an additional folding delay was observed. Because Gly missense mutations that cause disease are often located near natural interruptions, structural and folding perturbations arising from such proximity could be a factor in collagen genetic diseases.     

A new approach to the thermodynamic role of imino acids in collagen. Solution of a thermodynamic paradox]

The dependence of denaturation transition thermodynamic parameters in various collagens from imino acid compositions has been analysed. Computational and experimental data suggest independence of the collagen molecule hydration on imino acid composition and sequence in the polypeptide chain. The continuous net of hydrogen bonds is interrupted, if imino acid residues occur in the sequence of amino acid residues, as follows from Monte Carlo computations, because the hydrogen of NH-group plays sufficient role in water shell formation for this conformation. As a consequence, entropy of denatured collagen-water system increases hand by hand with increasing imino acid content and therefore delta S increases. The increase of enthalpy of transition from imino acid content is determined by favorable Van der Waals interactions of pyrrolidine rings in native triple helical collagen structure. It was pointed out that proline role is determined by decreasing hydration in the single stranded polypeptide chain in Polyproline II conformation that leads to an increase of entropy of the polypeptide-water system. Thus, the collagen structure formation by imino acids is promoted in the water media due to single chain left-helical conformation being unfavorable for proline residues as well as due to the enthalpy nature of the triple helix stabilization.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Identification of surface-exposed segments of apolipoprotein B-100 in the LDL particle

The isolation and amino acid sequence of eleven peptides liberated by tryptic treatment from surface-exposed regions of apolipoprotein B-100 in the native low-density lipoprotein particle are described. These peptides represent eight segments in the sequence of the B-100 protein, one of which was localised to the amino-terminal thrombolytic fragment T4 (1297 amino acids), four to the T3 fragment (2052 residues) and three to the carboxylterminal fragment T2 (1287 residues). An exposed segment was identified on each side of the T2/T3 cleavage site, in close proximity to two segments enriched in basic amino acids (residues 3147-3157 and 3359-3367 respectively). The surface exposure of this region is consistent with its contribution to the putative apo-B,E receptor binding domain. Four of the eight tryptic segments contribute to regions of proline-rich clusters. Homology between the sequence of the tryptic peptides and those predicted by cDNA cloning was complete.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

Amino acid sequence of the triple-helical domain of human collagen type VI

The complete amino acid sequence of the triple-helical domain of human collagen VI was deduced from sequences of appropriate cDNA clones and confirmed to about 50% by Edman degradation of tryptic peptides. This domain consists of three different peptide segments containing some 335-336 amino acid residues originating from central portions of the alpha 1 (VI), alpha 2(VI), and alpha 3(VI) chains, respectively. Sequence identity in the X/Y positions of the Gly-X-Y repeats is rather low (10-15%) between the chains. Peculiar features of these sequences include 3 cysteine residues about 50 (alpha 3(VI)) and 89 (alpha 1(VI), alpha 2(VI)) residues away from the N-terminus and several Gly-X-Y interruptions clustered in the C-terminal two-thirds of the triple helix. These structures are presumably required for cross-linking collagen VI oligomers and for super-coiling of triple helices in the dimers. Other features include 11 Arg-Gly-Asp sequences, some of which are likely to be used as cell-binding sites, and four Asn-X-Thr sequences, allowing N-linked glycosylation along the triple helix. Junctional areas close to the helix contain short, cysteine-rich segments which may seal the triple-helical domain through disulfide bond formation, endowing it with high stability. These features, together with a low sequence homology to fiber-forming and basement-membrane collagens, document the unique character of collagen VI, whose triple helix is specifically adjusted for forming microfibrils in tissues.     

Amino acid sequence of the N-terminal aggregation and cross-linking region (7S domain) of the alpha 1 (IV) chain of human basement membrane collagen

The amino acid sequence of the 216-residue-long N-terminal aggregation and cross-linking 7S domain of the alpha 1 (IV) chain of human placental basement membrane collagen is presented. The N terminus of the alpha 1 (IV) chain starts with a non-triple-helical region, which is at least 15 residues long and contains four cysteine and two lysine residues as putative cross-linking sites. This segment is followed by a 120-residue-long triple helical region, which contains the unusual occurrence of a cysteine residue in the Xaa position of a Gly-Xaa-Yaa triplet. Since individual molecules in the 7S domain are associated in an antiparallel manner, this cysteine probably aligns with one of the four cysteines in the amino-terminal end of an adjacent molecule, forming an intermolecular disulfide bridge. The length of the overlap of two adjacent molecules is estimated to be about 110 residues. The triple helix adjacent to the overlap zone is interrupted by a 10-residue-long non-helical area, which is probably responsible for the flexible region of the molecules in the neighbourhood of the overlap zone observed in the electron microscope. The mode of aggregation of the 7S domain, the formation of intermolecular cross-links as well as the relatively high stability of this region against proteolytic attack are discussed in the light of the elucidated amino acid sequence.