Evolutionary Origins of C-Terminal (GPP)n 3-Hydroxyproline Formation in Vertebrate Tendon Collagen

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)_n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)_n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)_n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)_n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)_n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.     

Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens

Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except α1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in α1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only α2(I) and α2(V) chains had any 3Hyp at the A3 site, whereas in chicken all α-chains except α1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus α1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.     

A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.     

Attaching human fibroblasts secrete a type I procollagen rich in 3-hydroxyproline.

The predominant collagenous protein secreted during the attachment of freshly trypsinized human foreskin fibroblasts was found to be Type I procollagen. Evidence is presented that both the α₁ and α₂ chains exhibit a 3-hydroxyproline/4-hydroxyproline ratio 4–5 fold higher than that of normal Type I collagen. These findings suggest that caution should be exercised in assigning an observed increase in the 3-hydroxyproline/4-hydroxyproline ratio to the synthesis of a basement membrane type collagen.     

Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro⁹⁸⁶ in A-clade chains α1(I), α1(II), and α2(V). Two partially modified sites (A2 and A3) were found at Pro⁹⁴⁴ in α1(II) and α2(V) and Pro⁷⁰⁷ in α2(I) and α2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro⁴⁷⁰ in α2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian α1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex.

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.     

Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly

Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.     

Increasing Effect of an Oral Intake of L-Hydroxyproline on the Soluble Collagen Content of Skin and Collagen Fragments in Rat Serum

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.     

Changes induced by ozone and ultraviolet light in type I collagen. Bovine Achilles tendon collagen versus rat tail tendon collagen

High-molecular-mass aggregates were made soluble from insoluble collagens of bovine Achilles tendon and rat tail tendon by limited thermal hydrolysis. These polymeric collagen aggregates were cross-linked by 390-nm-fluorescent 3-hydroxy-pyridinium residues (excited at 325 nm) in the former tendon and by unknown non-fluorescent residues in the latter. With the solubilized insoluble-collagens from both tendons, as well as with acid-soluble collagen from rat tail tendon, other 350-385-nm fluorescence intensities (excited at 300 nm) were found to be higher in monomeric chains than in dimeric and polymeric chains. Low levels of ozone inhibited fibril formation of acid-soluble collagen particularly from young rat tail tendon, reacting with tyrosine residues and the 350-385-nm fluorophores. Aldehyde groups, involved in cross-linking, were not effectively modified by ozone. beta-Components (alpha-chain dimers) were not efficiently dissociated even by higher doses of ozone compared to gamma-components (alpha-chain trimers). Polymeric chain aggregates from bovine Achilles tendon collagen, whose 3-hydroxy-pyridinium cross-links are cleaved by ozone, were more readily dissociated by ozone than those from rat tail tendon collagen. Ultraviolet (300-nm) light, which destroyed the 350-385-nm fluorophores, inhibited fibril formation less effectively than ultraviolet (275-nm) light, which is absorbed by tyrosine residues, and did not dissociate collagen polymers from rat tail tendon. On the other hand, ultraviolet (320-nm) light, absorbed by 3-hydroxy-pyridinium cross-links which were rapidly photolyzed, partially dissociated polymeric collagen aggregates from bovine Achilles tendon after subsequent heating.     

Collagen polymorphism: isolation and partial characterization of alpha 1(I)-trimer molecules in normal human skin.

Human skin has previously been shown to contain at least two genetically distinct types of collagen, type I and III. Here the presence of an additional form of collagen, α1(I)-trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 m NaCl at pH 7.4. The α-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 m NaCl precipitates contained predominantly type I collagen with a chain composition of [α1(I)]₂α2. In the 1.5 m precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 m NaCl fraction consisted almost exclusively of α-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with α1(I). The amino acid composition of these chains was also similar to that of α1(I), except that hydroxylysine was increased and lysine was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the α-chains in α1(I)-trimer are the same gene products as α1 in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in α1(I)-trimer. Estimation of the quantitative amounts of α1(I)-trimer indicated that this collagen accounts for less than 5% of the total collagen in adult human skin. It is speculated, however, that α1(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

The peptides acetyl-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 and acetyl-(Gly-Pro-3(S)Hyp)10-NH2 do not form a collagen triple helix

Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)-hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and 1H nuclear magnetic resonance spectroscopy. At 4 degrees C in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro-4(R)Hyp)10-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95 degrees C without an obvious transition. Although the peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 forms a trimer at 10 degrees C, sedimentation equilibrium experiments indicate that Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 is a monomer in water at 7 degrees C. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)10-NH2. This peptide also does not form a triple helix in water. 1H Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 nor Ac-(Gly-Pro-3(S)Hyp)10-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable -Gly-Pro-4(R)Hyperepeated host sequence, Ac-(Gly-Pro-4(R)Hyp)3-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms as stable a trimer (Tm=49.6 degrees C) as Ac-(Gly-Pro-4(R)Hyp)8-Gly-Gly-NH2 (Tm=48.9 degrees C). Given that Ac-(Gly-Pro-4(R)Hyp)3-Gly-4(R)Hyp-Pro-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (Tm=45.0 degrees C) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)10-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predictors of triple helical stability.     

Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.     

Crtap and p3h1 knock out zebrafish support defective collagen chaperoning as the cause of their osteogenesis imperfecta phenotype

Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients’ reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.     

Procollagen production by rat hepatocytes in primary culture

Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3–5 days and declined thereafter. The rate of collagen production was appox. one-tenth that observed by the rat skin fibroblasts of the same animals after 3–5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as α1(I) and α2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are nor major collagenous products of these cells.     

Transfection of normal human skin fibroblasts with human α1(I) and α2(I) collagen gene constructs and evidence for their coordinate expression

Type I collagen is composed of two α1(I) chains and one α2(I) chain that together form a unique triple helical structure. The genes for these chains are located on different chromosomes but their expression is tightly regulated. In order to investigate the mechanism of regulation of coordinate expression of these genes, I examined conditions for the efficient transfection of normal human skin fibroblasts with luciferase reporter gene constructs containing noncoding region of the first exon and the upper 500 base pairs sequence of the α1(I) or α2(I) gene. Expression ratio of these two reporter gene constructs was two to one, indicating these regions of α1(I) and α2(I) genes contain essential regulatory elements for the coordinate expression of α1(I) and α2(I) genes located on different chromosomes.     

Collagen in its fibrillar state is protected from glycation

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.     

Immunological cross-reactivity between electric-eel acetylcholinesterase and rat-tail-tendon collagen.

Immunological cross-reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels. 1. Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed-type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14-S + 18-S molecular forms). However, when the glubular 11-S enzyme was tested, almost no cross-reactivity was obtained. Similarly, guinea pigs immunized with 14-S + 18-S preparation exhibited skin sensitization to rat tail tendon collagen. 2. Using a radioimmunoassay, it was observed that 125I-labeled 14-S + 18-S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I-labeled 11-S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay. The experiments suggest that 14-S + 18-S acetylcholinesterase, but not 11-S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.     

The crystal structure of the collagen-like polypeptide (glycyl-4(R)-hydroxyprolyl-4(R)-hydroxyprolyl)9 at 1.55 A resolution shows up-puckering of the proline ring in the Xaa position

The collagen triple helix is characterized by the repeating sequence motif Gly-Xaa-Yaa, where Xaa and Yaa are typically proline and (2S,4R)-4-hydroxyproline (4(R)Hyp), respectively. Previous analyses have revealed that H-(Pro-4(R)Hyp-Gly)(10)-OH forms a stable triple helix, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not. Several theories have been put forth to explain the importance of proline puckering and conformation in triple helix formation; however, the details of how they affect triple helix stability are unknown. Underscoring this, we recently demonstrated that the polypeptide Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forms a triple helix that is more stable than Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). Here we report crystal the structure of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide at 1.55 A resolution. The puckering of the Yaa position 4(R)Hyp in this structure is up (Cgamma exo), as has been found in other collagen peptide structures. Notably, however, the 4(R)Hyp in the Xaa position also takes the up pucker, which is distinct from all other collagen structures. Regardless of the notable difference in the Xaa proline puckering, our structure still adopts a 7/2 superhelical symmetry similar to that observed in other collagen structures. Thus, the basis for the observed differences in the thermodynamic data of the triple helix<--> coil transition between our peptide and other triple helical peptides likely results from contributions from the unfolded state. Indeed, the unfolded state of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide seems to be stabilized by a preformed polyproline II helix in each strand, which could be explained by the presence of a unique repeating intra-strand water-mediated bridge observed in the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH structure, as well as a higher amount of trans peptide bonds.