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We have determined the amino acid sequence of the α chain of a fibril-forming collagen from the body wall of the marine invertebrate Riftia pachyptila (vestimentifera) by Edman degradation. The pepsin-solubilized collagen chain consists of a 1011-residue triple-helical domain and short remnants of N- and C-telopeptides. The triple-helical sequence showed one imperfection of the collagen Gly-Xaa-Yaa triplet repeat structure due to a Gly Ala substitution. This imperfection imperfection is correlated to a prominent kink in the molecule observed by electron microscopy. No strong sequence similarity was found with the fibril-forming vertebrate collagen types I-III, V and XI except for the invariant Gly residues. However, one of the two consensus cross-linking sequences was well conserved. The Riftia collagen shared with the vertebrate collagens many post-translational modifications. About 50% of the Pro and Lys residues are found in the Yaa position and were extensively hydroxylated to 4-hydroxyproline (4Hyp) and hydroxylysine (Hyl). A few proline residues in Xaa position were partially hydroxylated to either 4Hyp or 3Hyp. Despite the low sequence similarity, Riftia collagen was a potent adhesion substrate for two human cell lines. Cell adhesion could be inhibited by antibodies against the integrin β1 subunit but not by RGD peptides. This biological activity is apparently conserved in fibril-forming collagens of distantly related species but does not require the two RGD sequences present in Riftia collagen.

The collagen of a primitive invertebrate, the sea-pen Veretillum Cnidaria, Octocorallia), was studied with respect to its molecular-chain composition. The soft extracellular tissues (mesoglea) were solubilized by limited pepsin proteolysis and the collagen was isolated by selective precipitation at 0.7 M NaCl under acidic conditions. The pepsinized molecules were 260 nm in length, as demonstrated by electron microscope studies of rotary-shadowed molecules and of the segment-long-spacing crystallites obtained by dialysis against ATP. SDS/PAGE of the extract produced two main bands susceptible to bacterial collagenase, designated as the alpha 1 and alpha 2 chain, which were differentiated clearly by their CNBr cleavage products and the higher glycosylation rate of the alpha 2 chain. The latter finding corresponds with the high hydroxylysine content of the alpha 2 chain. The alpha 1/alpha 2 chain ratio observed in SDS/PAGE and the fact that only one peak was obtained by concanavalin-A affinity chromatography of a non-denatured 0.7 M NaCl extract demonstrate the alpha 1 [alpha 2]2 molecular structure of this collagen. These results contrast with data on the structure of other coelenterates (i.e. [alpha]3 for sea anemone collagen molecules and alpha 1 alpha 2 alpha 3 for jellyfish collagen molecules). They are discussed in relation to the evolution of collagen.The collagen of a primitive invertebrate, the sea-pen Veretillum Cnidaria, Octocorallia), was studied with respect to its molecular-chain composition. The soft extracellular tissues (mesoglea) were solubilized by limited pepsin proteolysis and the collagen was isolated by selective precipitation at 0.7 M NaCl under acidic conditions. The pepsinized molecules were 260 nm in length, as demonstrated by electron microscope studies of rotary-shadowed molecules and of the segment-long-spacing crystallites obtained by dialysis against ATP. SDS/PAGE of the extract produced two main bands susceptible to bacterial collagenase, designated as the alpha 1 and alpha 2 chain, which were differentiated clearly by their CNBr cleavage products and the higher glycosylation rate of the alpha 2 chain. The latter finding corresponds with the high hydroxylysine content of the alpha 2 chain. The alpha 1/alpha 2 chain ratio observed in SDS/PAGE and the fact that only one peak was obtained by concanavalin-A affinity chromatography of a non-denatured 0.7 M NaCl extract demonstrate the alpha 1 [alpha 2]2 molecular structure of this collagen. These results contrast with data on the structure of other coelenterates (i.e. [alpha]3 for sea anemone collagen molecules and alpha 1 alpha 2 alpha 3 for jellyfish collagen molecules). They are discussed in relation to the evolution of collagen.

Proteolytic digests of liver plasma-cell membranes from the cow were fractionated to yield two homogeneous glycopeptides and a third preparation about 92% pure. The composition of the two homogeneous glycopeptides made it clear that they were derived from basement membrane material rather than the plasma membrane. Ruminants are unusual in having large amounts of basement membrane in the liver while other animals generally have little or none. Both basement-membrane-derived glycopeptides contained a glucosyl galactosyl disaccharide linked to hydroxylysine, the smaller one contained no other sugar structure but the larger one contained in addition an acidic heterosaccharide, the two chains probably being linked separately to the same molecule. Smith degradation and β-elimination operations show that this heterosaccharide has an inner structure containing mannose and hexosamine, with the sugars galactose, N-glycollyl-neuraminic acid and fucose situated more peripherally. The amino-acid-hetero-saccharide linkage is alkali stable. The third glycopeptide, which may be plasma-membrane-derived, differs from the heterosaccharide saccharide described above in that it contains no glucose and contains some O-seryl and O-threonyl amino-acid-sugar linkage. It, too, has a periodate-resistant structure of hexosamine and mannose.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).

Metal activation of galactosylhydroxylysyl glucosyltransferase was studied with EDTA-treated highly purified enzyme from chick embryos and EDTA-treated citrate-soluble collagen substrate. Mn2+ activated the transferase at much lower concentrations than thought previously, and high Mn2+ concentrations were more effective than could be expected from the Michaelis-Menten equation. The data suggest that the enzyme can bind at least two Mn2+ ions, the first having a Kd of 3–5 μM (site I) and the second 50–70 μM (site II). The binding of the second Mn2+ decreases the Km of UDP-glucose from 25 μM to 6–7 μM, whereas the V at a saturating concentration of UDP-glucose is unaffected. The EDTA-treated enzyme possessed some activity even without added Mn2+, suggesting that either the transferase can be activated in the absence of any metal or that it has one additional metal binding site with high affinity. The kinetic data would be consistent with a reaction scheme involving an ordered binding of Mn2+, UDP-glucose and the collagen substrate at low Mn2+ and an ordered binding of Mn2+, Mn2+, UDP-glucose and the collagen substrate at high Mn2+ concentrations. In both cases the binding of the Mn2+, which occurs before that of UDP-glucose, is at thermodynamic equilibrium. Zn2+, Cu2+, Cd2+, Ni2+ and Ca2+ are inhibitors of the Mn2+-activated enzyme. Fe2+ (Kd 5–7 μM) and Co2+ (Kd 30 μM) also activate the enzyme at low concentrations, whereas no further activation, but instead an inhibition, was found at high concentrations. Ca2+ and Mg2+, which have previously been reported to serve as metal co-factors, did not activate the enzyme alone and did not stimulate the glucosyltransferase activated at low Mn2+ concentrations. The Kd values for Mn2+ at site II and for Co2+ are probably much higher than the physiological concentrations, whereas the activation by Mn2+ at site I and by Fe2+ may be of physiological significance.

The hydroxylsine-linked disaccharide unit, 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosyl-hydroxylysine (Glc-Gal-Hyl), prepared from collagens, was hydrolyzed by a glucohydrolase present in rat spleens and lungs. This disaccharide unit was scarcely hydrolyzed by homogenates of intestines, livers, and kidneys, which had a high alpha-D-glucosidase activity for neutral glucosides. The Glc-Gal-Hyl glucohydrolase was purified from rat spleens by affinity chromatography and gel filtration to the extent that sodium dodecyl sulfate/polyacrylamide gel electrophoresis gave a single band stained by Coomassie blue G-250. This purified glucohydrolase had a pH optimum around 5.8, and the Michaelis constant was 5.9 mM when Glc-Gal-Hyl was used as a substrate. This enzyme did not hydrolyze neutral glucosides. It is concluded that this Glc-Gal-Hyl glucohydrolase is responsible for catabolism of the hydroxylsine-linked disaccharide unit derived from collagens in mammals.

Calf corneas were extracted with a buffer containing M CaCl2, 0.05 M Tris and 0.02 M citric acid, at pH 7.5. The residual stroma was digested successively by collagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. The sugar and amino acid content of the isolated glycopeptides was determined. Two types of sugar chains were detected: (a) chains with a more complicated structure, containing galactose, glucose, mannose and glucosamine; (b) disaccharidic and monosaccharidic side chains containing galactose and glucose or galactose alone and linked O-glycosidically to the OH groups of hydroxylysine. The Hyl-Gal-Glc compound was isolated and identified with the compound isolated by Butler and Cunningham from calf skin collagen, by the study of its composition, thin layer chromatography and high voltage electrophoresis. The stroma of the calf cornea contains about 4 times more hexoses in the small glycopeptides of type b (di- and monosaccharides) than in the higher molecular weight mannose-containing chains.

The distribution of the heteropolysaccharide chains in the insoluble tissular network of the dogfish cornea was investigated. For this purpose, the corneas were extracted with a buffer solution composed of 1 M CaCl2, 0.05 M tris and 0.02 M citric acid to remove soluble proteins. The residual insoluble stroma was successively hydrolysed by collagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. Two types of sugar chains were detected: (a) polyosides composed of galactose, glucose, mannose, glucosamine, fucose, xylose and sialic acid, some of them at least linked to aspartic acid (type 1); (b) galactose or glucosidogalactose linked to hydroxylysine (type 2). For one heteropolysaccharide chain of type 1, 6 galactosidohydroxylysines and 4 glucosidogalactosidohydroxylysines were found. The insoluble stroma was submitted to a fractionated extraction procedure by trichloroacetic acid and water in order to remove all collagenous material. The residual structural glycoprotein exempt from hydroxyproline was hydrolysed by pronase and the glycopeptide fraction was isolated by exclusion chromatography and electrophoresis. A single major glycopeptide fraction of the type 1 containing galactose, glucose, mannose, xylose and glucosamine was obtained. This clear-cut separation of the two types of glycopeptides by the tricholoroacetic acid treatment indicates that they are bound to different peptide chains: glycane of type 1 is bound to the peptide chain of structural glycoproteins (insoluble in hot trichloroacetic acid) and the glycane of type 2 is bound to the peptide chains of collagen (soluble in hot trichloroacetic acid).

A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistance towards further degradation by bacterial collagenase at 20 degrees C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360,000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm = 48 degrees C and 70 degrees C. The more stable domain could be isolated as an homogeneous fragment (Mr = 225,000) after a second digestion with collagenase at 37 degrees C. This fragment contained all the disulfide bonds (42 Cys/1,000 residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 X 95 nm (long form) and 2.4 X 40-50 nm (short form). Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27,000-153,000 which differ from each other by Mr increments 25,000-27,000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transiton of the triple helix to 48 degrees C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 degrees C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.

A collagenous trimeric cross-linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation-exchange chromatography and reversed-phase HPLC. Molecular mass and amino acid composition indicated that the C-terminal, non-helical region of type I collagen in its dimer form, designated as [ColC(I)]2, is cross-linked to a tryptic peptide TN(I) from the N-terminal helical cross-link region of an adjacent type I molecule, forming the cross-linked peptide [ColC(I)]2 X TN(I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross-linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers. The trifunctional cross-linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three-chain structure. Pyridinoline was isolated from the cross-linked peptide by preparative amino acid analysis and reversed-phase HPLC and identified by its ultraviolet absorption spectra, its fluorescence excitation and emission spectra and, for the first time, its time-of-flight secondary ion-mass spectrum. The high sensitivity of the latter method, exceeding that of fast-atom-bombardment mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range. The occurrence of pyridinoline in non-stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross-linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross-link form that is different from pyridinoline and is hydrolysable.

1. Desmosine and isodesmosine were detected in an invertebrate molluscan species, i.e. in an insoluble protein in the hinge ligament of a bivalve species, Sakhalin surf clam (Pseudocardium sachalinensis, in family Mactridae). The protein is rich in glycine and methionine S-oxide but devoid of hydroxyproline and hydroxylysine. 2. 3,3′-Methylenebistyrosine was also detected in the HCl hydrolysate of the hinge-ligament protein, but it was found to be an artefact produced from tyrosine and formaldehyde derived from methionine S-oxide during the HCl hydrolysis of the protein.

Segment-long-spacing crystallites and reconstituted fibrils have been made from collagen extracted from the ejaculatory duct of the adult male locust, Locusta migratoria. These show the same banding pattern after positive staining as segment-long-spacing crystallites and fibrils made from mammalian type I collagen. Native fibrils show the same periodic pattern as type I fibrils, but it is not so distinct. Biochemical analysis of pepsin-digested locust collagen shows that there are two collagenous components. The alpha chains of the major component are similar to mammalian alpha 1 (I) chains, except that the number of hydroxylysine residues is elevated and the CNBr peptides differ. There are no alpha 2 chains; hence this locust collagen molecule is an alpha 1 trimer. The second component, which is present in only minute quantities, may be a type IV basement membrane collagen. It is concluded that the fibrous collagen molecule of the locust is very similar to that of mammalian type I trimers and to those of other invertebrates which have been examined.

The amino acid compositions of Metridium-, Lumbricus- and Mytilus-collagens corresponded to those of the lowest vertebrates: the content of imino acids was low and that of serine and threonine high. Metridium-collagen also contained cystine and large amounts of hydroxylysine. In Lumbricus-collagen the hydroxylation of proline was almost complete and the total content of hydroxy amino acids thus exceptionally high. The solubility of collagens from the invertebrates varied in wide range. In some cases, e. g. the byssus apparatus of Mytilus, the insolubility seems to depend on the stabilizing effect of the adjacent materials which is manifested also in the unique thermal shrinking temperature of 90°. The starch-gel electrophoretic patterns were studied on soluble collagens from the following species: octopus, Loligo, Lumbricus, Diphyllobothrium, Mytilus, and Metridium. A single α-component pattern, resembling that from lamprey, was observed in all the cases.

1 Chick embryo skin slices were incubated with [14C]proline without and in the presence of 1 mM 2,2′-dipyridyl. Collagen was isolated and digested with collagenase. The kinetics of the splitting off of small radioactive fragments showed a more rapid degradation of the undenatured hydroxyproline and hydroxylysine deficient collagen which had been formed in the presence of 2,2′-dipyridyl, compared to the control sample. No differences in the rate of digestion have been found between heat denatured control and atypical collagen. 2 In parallel experiments the rate of collagenase degradation of proline rich Ascaris cuticle collagen was several times greater than that of calf skin collagen. 3 Skin slices incubated simultaneously with [14C]glutamic acid and 0.5 mM dipridyl showed more radioactive proline than did control samples. This is further evidence for increased degradation of atypical collagen because under these experimental conditions the metabolism of non-collagenous protein was the same in control and dipyridyl treated samples. It is apparent that hydroxyproline and hydroxylysine decicient collagen is more susceptible to the action of collagenase and other tissue proeases.

Long spacing segment crystallites, prepared from soluble chick bone and skin collagen with the chain composition [α1(I)]2α2 and soluble chick cartilage collagen with a chain composition [α1(II)]3, were compared in electron microscope studies after staining with phosphotungstic acid and uranyl acetate. Although comparison of bone and skin collagen long-spacing segment preparations with similar preparations of cartilage collagen revealed several differences in intensities of cross-striation bands, no differences in the positions of the cross-striation bands could be observed in the three preparations, indicating that the distribution of polar and apolar regions along the cartilage collagen molecule is markedly similar to that of molecules derived from bone and skin collagen. Comparison of segment long-spacing crystallites prepared from the various collagens also showed that the cartilage collagen is longer than the bone or skin collagen molecule by about 10–12 nm. The additional length of the cartilage collagen molecule is clearly discerned as an extra band located in the COOH-terminal region, and is removed by treatment of the collagen with pepsin prior to precipitation as segment long-spacing crystallites, indicating that this region contains one or more sequences in which the collagen helix cannot form. In studies on fibrils formed by the three collagen preparations, it was noted that bone and skin collagen could be readily precipitated as broad, well-structured fibrils in which a banding pattern characteristic of native type fibrils could be easily observed. On the other hand, cartilage collagen, precipitated under identical conditions, formed slightly smaller fibrils in which a banding pattern commonly associated with native type collagen fibrils was only faintly visible in certain segments of the fibrils. These results suggest that the relatively large number of bulky carbohydrate prosthetic groups covalently attached to hydroxylysine residues in the α1(II) chains of cartilage collagen sterically hinder the deposition of staining materials and thus prevent the clear manifestation of the banding pattern in fibrils of cartilage collagen.

A method involving the use of sieves and differential centrifugation for the preparation and separation of basement membranes from human renal tubules and glomeruli is described. Electron microscopic examination of the material obtained shows the glomerular membrane as a single dense structure, while the tubular membrane is composed of two dense layers separated by a space filled with loose fibrils exhibiting the same periodicity as collagen. The carbohydrate composition of both types of membranes is the same but tubular membranes have a lower content of some specific sugars. Differences also exist in amino acid composition. The content in hydroxylysine is higher in the glomerular membrane while the content in hydroxyproline is higher in the tubular membrane. Gel electrophoresis after solubilization of the material demonstrates the presence of two, may be three, major proteins.