A second fibronectin-binding region is present in collagen alpha chains

The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.     

Amino acid sequence of the beta chain of human fibrinogen.

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.     

The identity of a cyanogen bromide fragment of bovine dentin collagen containing the site of an intermolecular cross-link.

A peptide fraction isolated from a cyanogen bromide digest of bovine dentin collagen had a molecular weight of 46000. Its size and amino acid composition indicated that it could not consist of peptides derived from the cleavage of a single alpha chain. On reduction with tritiated sodium borohydride, radioactivity was incorporated primarily into 5, 5'-dihydroxylysinonorleucine without degradation at the peptide backbone. Periodate cleavage of the reduced or nonreduced peptide fraction generated one fragment of molecular weight 28000 and one of 18000 completely accounting for the size of the parent peptide. On amino acid analysis the constituent single-chain peptides were determined to be alpha2CB4 and alpha1CB6. Both peptides isolated after periodate oxidation of the tritiated borohydride reduced cross-link peptide were found to contain (3H)hydroxynorvaline. These data show that some hydroxylysine of alpha2CB4, a helical region peptide, was present in aldehyde form and could act as the aldehyde donor icross-link, Schiff's base formation. The only cross-linkage of this alpha2CB4 acting as an aldehyde donor peptide to alpha1CB6 would be a helical region to helical region bond, perhaps accounting for the unusual stability and low solubility of dentin collagen.     

Substitution of cysteine for glycine within the carboxyl-terminal telopeptide of the alpha 1 chain of type I collagen produces mild osteogenesis imperfecta

We have characterized a mutation that produces mild, dominantly inherited osteogenesis imperfecta. Half of the alpha 1 (I) chains of type I collagen synthesized by cells from an affected individual contain a cysteine residue in the 196-residue carboxyl-terminal cyanogen bromide peptide of the triple-helical domain (Steinmann, B., Nicholls, A., and Pope, F. M. (1986) J. Biol. Chem. 261, 8958-8964). Unexpectedly, sequence determined from a proteolytic fragment of the alpha 1 (I) chain derived from procollagen molecules synthesized in the presence of both [3H]proline and [35S]cysteine indicated that the cysteine is located at the third residue carboxyl-terminal to the triple-helical domain, normally a glycine. The nucleotide sequence of a fragment amplified from genomic DNA confirmed the location of the cysteine residue and showed that the mutation was a single nucleotide change in one COL1A1 allele. This represents a new class of mutations, point mutations outside the triple-helical domain of the chains of type I collagen, that produce the osteogenesis imperfecta phenotype.     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.     

Patterns of hemoglobin assembly in reticulocytes of sickle cell trait individuals.

Venous blood was obtained from five sickle cell trait donors with relatively high hemoglobin S concentrations (40% of total hemoglobin) and five donors with unusually low hemoglobin S concentrations (25 to 30%). A fraction of cells with 15 to 20% reticulocytes was isolated from the blood and incubated with [3H]leucine in a medium supporting protein synthesis for various times from 1.25 to 60 min. Previous studies showed an imbalance in globin chain synthesis in reticulocytes of "low hemoglobin S" donors which suggested the presence of an alpha-thalassemia gene; reticulocytes of "high hemoglobin S" donors had balanced globin chain synthesis (DeSimone, J., Kleve, L., Longley, M.A., and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 59, 564-569). In the present study the soluble phase of the 3H-labeled reticulocytes was examined by electrophoresis on strips of cellulose acetate. The tetramer hemoglobins A and S were separated from each other and from a small pool of free, newly synthesized alpha and beta chains. Kinetics of labeling studies showed that the free alpha and beta chains were intermediates in tetramer hemoglobin assembly. The distribution of radioactivity between the alpha and beta chains of each of the electrophoretically isolated components were determined by separation of their globin chains on CM-cellulose columns. After 5 min of 3H-labeling of the reticulocytes from donors with 40% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the tetramer hemoglobins A and S ranged from 0.37 to 0.58. This ratio increased with longer labeling times. Almost all of the radioactivity of the free chain intermediates was in the alpha chain. These results confirmed the presence of a significant pool of newly synthesized alpha chains and a normal pattern of hemoglobin assembly in which initially unlabeled alpha chains combined with labeled beta chains when the cells were exposed to [3H]leucine. Conversely, in the reticulocytes of donors with 25 to 30% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the completed hemoglobins A and S ranged from 0.96 to 1.37 and remained unchanged throughout the 3H-labelling period. The radioactivity of the free alpha chain pool was substantially less that the total radioactivity of the betaA and betaS chain pools. These results confirmed the existence of a decreased pool size of soluble alpha chain intermediates and a pattern of hemoglobin assembly consistent with the presence of the alpha-thalassemia gene.     

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.     

Hydroxylation of prolyl residues in type II procollagen in vitro and in cellulo. Lack of preferential hydroxylation of specific regions of the protein

[14C]Proline-labeled protocollagen, the unhydroxylated form of procollagen, was isolated from cartilage cells incubated with alpha, alpha'-dipyridyl. For examination of the initial steps in the hydroxylation of the protein, it was incubated in vitro with prolyl hydroxylase so that an average of 1.3-2.7 prolyl residues per chain was hydroxylated. The partially hydroxylated alpha chain were cleaved with cyanogen bromide, and the fragments were separated by polyacrylamide gel electrophoresis or column chromatography. The cyanogen bromide fragments were hydroxylated to the same degree. The results indicated, therefore, that in the initial hydroxylation of alpha chains in vitro, there was no preferential hydroxylation of any specific regions of the protein. In a second series of experiments, cartilage cells were incubated with [14C]proline and alpha, alpha'-dipyridyl so that prolyl hydroxylase in the cells was extensively, but not completely, inhibited. Partially hydroxylated alpha chains were isolated, and cyanogen bromide fragments of the alpha chains from the cells were assayed for hydroxy[14C]proline. The alpha chains contained an average of two residues of hydroxyproline per chain, and the cyanogen bromide fragments were hydroxylated to about the same degree. The results indicated, therefore, that when prolyl hydroxylase activity in cells is low relative to the rate at which pro alpha chains are synthesized, hydroxylation of prolyl residues occurs as it does in vitro, and there is no preferential hydroxylation of a specific region of the protein.     

Primary structure of human fibrinogen and fibrin. Structural studies on NH2-terminal part of B beta chain.

The cyanogen bromide fragment, N-DSK, containing the NH2-terminal portions of the three chains of fibrinogen, was found to exist in dimeric and polymeric forms. These different forms gave rise to identical chain fragments on reduction and alkylation. The B beta chain of N-DSK from fibrinogen and the beta chain of N-DSK from fibrin were isolated and characterized. The B beta chain fragment has a blocked NH2-terminal residue, and fibrinopeptide B is released on digestion with thrombin. The beta chain fragment has glycine as NH2-terminal residue. The molecular weight of the B beta chain fragment is 12200 as determined by ultracentrifugal analysis. Gel electrophoresis in sodium dodecyl sulphate gave the molecular weights of 14000 and 13000 for the B beta chain and beta chain fragments, respectively. The NH2-terminal B beta chain fragment consists of 118 amino acid residues and the beta chain fragment of 104 residues. The amino acid sequence of beta chain fragment is identical to B beta chain fragment except for the fibrinopeptide B portion. The isolation of a B beta-related fragment (B beta +), with a molecular weight of 30000, is also reported. The presence of B beta + was explained on the basis of incomplete cleavage at the Met-118 residue during treatment with cyanogen bromide. Some functional aspects of the B beta chain fragment are discussed.     

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.     

Sulfation of a tyrosine residue in the plasmin-binding domain of alpha 2-antiplasmin

Sulfation of human alpha 2-antiplasmin, the major plasma inhibitor of fibrinolysis, was examined using both protein isolated from human plasma and protein synthesized and biosynthetically labeled with [35S]sulfate by a human hepatoma-derived cell line. Linkage of sulfate to tyrosine was demonstrated by recovery of labeled tyrosine sulfate after base hydrolysis of sulfate-labeled alpha 2-antiplasmin. Analysis by reverse-phase high performance liquid chromatography of peptides released from alpha 2-antiplasmin by cleavage with trypsin or cyanogen bromide indicated that sulfate is linked to a single segment of the protein. A cyanogen bromide peptide corresponding to the sulfate-labeled peptide was prepared from alpha 2-antiplasmin isolated from human plasma. Consistent with the presence of tyrosine sulfate in this peptide, its chromatographic elution was altered by treatment with acid under conditions which release sulfate from a tyrosine residue. No peptide in the total digest of alpha 2-antiplasmin by cyanogen bromide eluted at the position of the peptide following desulfation, suggesting that all of the protein is in a sulfated form. The sequence of the sulfate-containing cyanogen bromide peptide as determined by sequential Edman degradation, amino acid composition, and fast atom-bombardment-mass spectrometry was: Glu-Glu-Asp-Tyr(SO4)-Pro-Gln-Phe-Gly-Ser-Pro-Lys-COOH. This peptide is a segment of the previously identified plasmin-binding domain of alpha 2-antiplasmin.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

Precursor sequence of MOPC-315 mouse immunoglobulin heavy and light chains.

Partially purified mRNA coding for the MOPC-315 heavy (alpha) or light (lambda 2) immunoglobulin chain was translated in a nuclease-treated reticulocyte lysate containing 20 labeled amino acids. Radiolabeled precursor heavy and light chains, purified by immunoprecipitation and preparative gel electrophoresis, were subjected to Edman degradation. The labeled phenylthiohydantoin derivatives obtained in each degradative cycle were identified and quantitated by high pressure liquid chromatography. Both heavy and light chain precursor segments were hydrophobic in nature; however, they were not homolgous in sequence. To establish whether COOH-terminal proteolytic processing of the heavy chain might also be occurring during secretion, the cyanogen bromide peptides of the heavy chain precursor were compared to those of the mature secreted heavy chain. The results indicated that the COOH termini of the two chains were identical.     

An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor

Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.     

The collagen of heart valve.

A hydroxylysine-rich type I collagen has been isolated from pepsin-digested porcine heart valve. The ratio of alpha1 to alpha2 in the isolated molecule was 2:1. The component alpha chains exhibited unusual chromatographic behavior in comparison to corresponding chains from human dermis and lathyritic rat skin collagen. The composition of component cyanogen bromide peptides identified the alpha chains as authentic type I chains and demonstrated hydroxylysine enrichment throughout the length of the chain. delta6-Dehydro-5,5'dihydroxylysinonorleucine, a collagen cross-link derived from two hydroxylysyl residues and ordinarily found in hard tissue collagens was found to be the predominant cross-link in heart valve.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.     

Identification of amino acid residues photolabeled with 2-azido[alpha-32P]adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase

When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido[alpha-32P]adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled [Dalbon, P., Boulay, F., & Vignais, P. V. (1985) FEBS Lett. 180, 212-218]. The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.     

The staining of type V collagen obtained from FBJ virus-induced osteosarcoma with concanavalin A

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.