The cloning and sequencing of alpha 1(VIII) collagen cDNAs demonstrate that type VIII collagen is a short chain collagen and contains triple-helical and carboxyl-terminal non-triple-helical domains similar to those of type X collagen

We have isolated two overlapping cDNA clones covering 2425 base pairs encoding a short type VIII collagen chain synthesized by rabbit corneal endothelial cells. The cDNAs encode an open reading frame of 744 amino acid residues containing a triple-helical domain of 454 residues flanked by 117- and 173-residue amino and carboxyl non-triple-helical domains (called NC2 and NC1, respectively). Based on the identity between the DNA-derived amino acid sequence and the amino acid sequence of a type VIII collagen CNBr peptide obtained from rabbit corneal Descemet's membrane, we conclude that the cDNAs code for a type VIII collagen chain. We give this chain the designation alpha 1(VIII). The alpha 1(VIII) triple-helical domain contains eight imperfections in the Gly-X-Y repeated structure with Gly-X instead of a full triplet. The length of the triple-helical domain and number and relative locations of these imperfections are remarkably similar to those of chicken alpha 1(X) collagen. The amino acid sequence of the carboxyl three-quarters of the NC1 domain has high sequence similarity to that of alpha 1(X) collagen. These data suggest that the triple-helix coding portions and carboxyl three-quarters of the NC1 domains of the alpha 1(VIII) and alpha 1(X) genes have a common evolutionary origin.     

Isolation and characterization of pepsin-solubilized human basement membrane (type IV) collagen peptides

Native type IV collagen was isolated from human placental tissue by pepsin digestion, fractional salt precipitation, reduction and alkylation, a second pepsin digestion, and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. After denaturation, 10 distinct peptides were isolated from this material by molecular sieve, ion-exchange, and high-performance liquid chromatography. All of the peptides were found to have amino acid compositions characteristic of type IV collagen. Analysis of the eight major peptides by amino-terminal amino acid sequencing and by cyanogen bromide and tryptic peptide mapping has revealed the manner in which they are derived from type IV collagen. Pepsin liberates two large peptides by attacking non-triple-helical regions, one derived from the alpha 1 (IV) chain (F2, Mr 90 000) and one derived from the alpha 2 (IV) chain (F3, Mr 75 000). The alpha 1 (IV)-derived F2 peptide is also represented in the pepsin digest by amino-terminal and carboxy-terminal subfragments [F4c (Mr 41 000) and F4a (Mr 60 000)], as is the alpha 2 (IV)-derived F3 peptide [F5 (Mr 28 000) and F4b (Mr 50 000), respectively]. These findings indicate that the molecular regions from which the larger peptides are derived in themselves contain pepsin-sensitive (non-triple-helical) domains. In addition, several of the peptides examined were found to be present in two slightly different forms, suggesting that closely adjacent pepsin-sensitive sites often exist within the type IV collagen molecules. The methods outlined here provide a reliable means by which identifiable type IV collagen peptides can be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

Type VIII collagen from bovine Descemet's membrane: structural characterization of a triple-helical domain

Bovine corneal Descemet's membrane (DM) was subjected to limited pepsin digestion. Soluble native collagens were fractionated by differential salt precipitation, and a mixture of type V collagen and collagenous fragments with a chain Mr of 50,000 (50K) was obtained at a concentration of 1.5 M NaCl. Further purification of the 50K collagen by molecular sieve and high-performance liquid chromatography resulted in the isolation of two-non-disulfide-bonded polypeptides, 50K-A and 50K-B, which were susceptible to several neutral proteases, including bacterial collagenase. By the criteria of peptide mapping, amino acid composition, and N-terminal sequence analysis, 50K-A and 50K-B were structurally dissimilar, although both chains contained Gly-X-Y repeats. 50K-A and 50K-B were immunologically and structurally distinct from collagen type I, III, IV, V, and VI. Immunohistochemical studies of bovine ocular tissue showed preferential distribution of the collagen containing the 50K fragment in the DM, with a more disperse arrangement of apparently interconnecting fibrils in the corneal stroma. Type VIII collagen isolated from the culture medium of metabolically radiolabeled bovine corneal endothelial (BCE) cells and its pepsin-resistant Mr 50 000 domain(s) both cross-reacted with antisera to 50K polypeptides from the corneal DM. Additionally, the CNBr peptide maps of pepsin-resistant Mr 50 000 polypeptides of type VIII collagen isolated from BCE cells and bovine corneal DM were highly similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.     

Structural characterization of a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A soluble glycoprotein of Mr = 80,000 has been isolated from lung lavage of patients with alveolar proteinosis and found to contain 5 residues of hydroxyproline, 91 residues of glycine, 3 residues of methionine, 3.8 molecules of sialic acid, 6 molecules of mannose, 5.9 molecules of galactose, 1 molecule of fucose, and 9.1 molecules of glucosamine. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted in four peptides with molecular weights of 36,000, 27,000, 12,000, and 5,000. The chemical compositions of the CNBr peptides indicated the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contained carbohydrate. Limited trypsin digestion of the glycoprotein of Mr = 80,000 resulted in four peptides with molecular weights of 62,000, 36,000, 26,000 and 18,000, the latter being the NH2-terminal peptide of the native glycoprotein molecule. The peptide of Mr = 26,000 was found to be the COOH-terminal peptide.     

The alpha 1 (VIII) collagen gene is homologous to the alpha 1 (X) collagen gene and contains a large exon encoding the entire triple helical and carboxyl-terminal non-triple helical domains of the alpha 1 (VIII) polypeptide

We recently cloned and sequenced alpha 1 (VIII) collagen cDNAs and demonstrated that type VIII collagen is a short-chain collagen that contains both triple helical and carboxyl-terminal non-triple helical domains similar to those of type X collagen (Yamaguchi, N., Benya, P., van der Rest, M., and Ninomiya, Y. (1989) J. Biol. Chem. 264, 16022-16029). We report here on the structural organization of the gene encoding the rabbit alpha 1 (VIII) collagen chain. The alpha 1 (VIII) gene contains four exons, whose sizes are 69, 120, 331, and 2278 base pairs. The first and second exons encode only 5'-untranslated sequences, whereas the third exon codes for a very short (3 nucleotides) stretch of 5'-untranslated sequence, the signal peptide, and almost the entire amino-terminal non-triple helical (NC2) domain (109 1/3 codons). Interestingly, the last exon encodes the rest of the translated region, including 7 2/3 codons of the NC2 domains, the complete triple helical domain (COL1, 454 amino acid residues), the entire carboxyl-terminal non-triple helical domain (NC1, 173 amino acid residues), and the 3'-untranslated region. This exon-intron structure is in stark contrast to the multi-exon structure of the fibrillar collagen (types I, II, III, V, and XI) genes, but it is remarkably similar to that of the type X collagen gene (LuValle, P., Ninomiya, Y., Rosenblum, N. D., and Olsen, B. R. (1988) J. Biol. Chem. 263, 18278-18385). The data suggest that the alpha 1 (VIII) and the alpha 1 (X) genes belong to the same subclass within the collagen family and that they arose from a common evolutionary precursor.     

Type VIII collagen: heterotrimeric chain association

Two chains, alpha1(VIII) and alpha2(VIII), have been described for type VIII collagen. Early work suggested that these chains were present in a 2:1 ratio, although recent work has shown that homotrimers can form and predominate in some tissues. In order to address the question of whether the alpha1(VIII) and alpha2(VIII) chains could co-polymerise we made a shortened alpha1(VIII) chain and expressed this with full length alpha2(VIII) chain in an in vitro translation system supplemented with semi-permeabilised cells. Heterotrimers containing either two or one alpha2(VIII) were evident. Interestingly, a point mutation in the NC1 domain of the alpha1(VIII) chain abrogated trimer formation. In addition we were able to demonstrate chain association of the alpha1(X) chain of type X collagen with the shortened alpha1(VIII) chain. Variations in chain association were seen when altered ratios of message were used. These results demonstrate the importance of the NC1 domain in chain association and suggest that gene expression regulates the composition and function of type VIII collagen by varying chain composition.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

Nonenzymatic glycation of type I collagen. The effects of aging on preferential glycation sites.

The present study was designed to investigate the effects of aging on preferential sites of glucose adduct formation on type I collagen chains. Two CNBr peptides, one from each type of chain in the type I tropocollagen molecule, were investigated in detail: alpha 1(I)CB3 and alpha 2CB3-5. Together these peptides comprise approximately 25% of the total tropocollagen molecule. The CNBr peptides were purified from rat tail tendon, obtained from animals aged 6, 18, and 36 months, by ion exchange chromatography, gel filtration, and high-performance liquid chromatography (HPLC). Sugar adducts were radiolabeled by reduction with NaB3H4. Glycated tryptic peptides were prepared from tryptic digests of alpha 2CB3-5 and alpha 1(I)CB3 by boronate affinity chromatography and HPLC. Peptides were identified by sequencing and by compositional analysis. Preferential sites of glycation were observed in both CB3 and alpha 2CB3-5. Of the 5 lysine residues in CB3, Lys-434 was the favored glycation site. Of the 18 lysine residues and 1 hydroxylysine residue in alpha 2CB3-5, 3 residues (Lys-453, Lys-479, and Lys-924) contained more than 80% of the glucose adducts on the peptide. Preferential glycation sites were highly conserved with aging. In collagen that had been glycated in vitro, the relative distribution of glucose adducts in old animals differed from that of young animals. In vitro experiments suggest that primary structure is the major determinant of preferential glycation sites but that higher order structure may influence the relative distribution of glucose adducts among these preferred sites.     

Type VIII collagen has a restricted distribution in specialized extracellular matrices

A pepsin-resistant triple helical domain (chain 50,000 Mr) of type VIII collagen was isolated from bovine corneal Descemet's membrane and used as an immunogen for the production of mAbs. An antibody was selected for biochemical and tissue immunofluorescence studies which reacted both with Descemet's membrane and with type VIII collagen 50,000-Mr polypeptides by competition ELISA and immunoblotting. This antibody exhibited no crossreactivity with collagen types I-VI by competition ELISA. The mAb specifically precipitated a high molecular mass component of type VIII collagen (EC2, of chain 125,000 Mr) from the culture medium of subconfluent bovine corneal endothelial cells metabolically labeled for 24 h. In contrast, confluent cells in the presence of FCS and isotope for 7 d secreted a collagenous component of chain 60,000 Mr that did not react with the anti-type VIII collagen IgG. Type VIII collagen therefore appears to be synthesized as a discontinuous triple helical molecule with a predominant chain 125,000 Mr by subconfluent, proliferating cells in culture. Immunofluorescence studies with the mAb showed that type VIII collagen was deposited as fibrils in the extracellular matrix of corneal endothelial cells. In the fetal calf, type VIII collagen was absent from basement membranes and was found in a limited number of tissues. In addition to the linear staining pattern observed in the Descemet's membrane, type VIII collagen was found in highly fibrillar arrays in the ocular sclera, in the meninges surrounding brain, spinal cord, and optic nerve, and in periosteum and perichondrium. Fine fibrils were evident in the white matter of spinal cord, whereas a more generalized staining was apparent in the matrices of cartilage and bone. Despite attempts to unmask the epitope, type VIII collagen was not found in aorta, kidney, lung, liver, skin, and ligament. We conclude that this unusual collagen is a component of certain specialized extracellular matrices, several of which are derived from the neural crest.     

Proteins of the periodontium. Identification of collagens with the [alpha1(I)]2alpha2 and [alpha1(III)]3 structures in bovine periodontal ligament.

Insoluble collagen was prepared from bovine periodontal ligament. Isolation and characterization of CNBr peptides originating from the alpha1(I), alpha2, and alpha1(III) chains showed that the tissue contained both type I and type III collagens. Further evidence for the presence of type III collagen was obtained by the isolation of alpha1(III) chains from pepsin-treated ligament collagen, with properties similar to those of human alpha1(III) chains. Estimates based on the amounts of certain CNBr peptides indicated that about one-fifth of the collagen of periodontal ligament is type III, the remainder being type I collagen.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Microquantitation of insoluble tissue collagen (types I and III) by radiodilution assay

The individual collagen types of the extracellular matrix of small tissue samples have been difficult to quantitate accurately both due to their marked insolubility and their relatively low immunogenicity. Thus no microassay with the sensitivity of a radioimmunoassay is currently available for quantitation of insoluble collagen types I and III in extremely small tissue samples. A radiochemical assay has been developed which allows direct processing of small tissue samples containing as little as 1-3 micrograms of a given collagen alpha chain. Unprocessed lyophilized tissues were digested with cyanogen bromide (CNBr) in the presence of a tritiated probe containing a soluble mixture of 3H-alpha 1(I) and 3H-alpha 1(III) collagen previously extracted and purified from tissue minces incubated with [3H]leucine. The resulting mix of radiolabeled peptides was separated on sodium dodecyl sulfate-polyacrylamide gradient gels. Reduction of the specific radioactivity of free leucine in acid hydrolysates of each individual CNBr peptide can be used to quantitate the amount of collagen types I or III in the original sample. Similar radiodilution analysis using a 3H-alpha 2(I) probe indicated a normal 2:1 ratio of alpha chains of type I collagen in the tissues tested. This method is also applicable to cell culture, easily measuring the collagen associated with fibroblast cell layers or medium in individual microtiter wells. When applied to various tissues of known collagen-type composition, it provides reproducible results which compare well with values published in the literature.     

The order of cyanogen-bromide peptides and location of carbohydrate in the alpha2 chain of guinea-pig skin collagen.

The alpha2 chain of guinea pig skin collagen contains two additional methionyl residues in comparison with the alpha2 chain of other vertebrate species. The order of the three CNBr peptides unique to the alpha2 chain was established on the basis of the homology of their primary structures to sequences of previously ordered regions in the alpha1 and alpha2 chains of other colagens. The two larger peptides, 4A + 4B, were found to correspond to the region homologous to alpha2-CB4 of other species, while the smaller peptide, 3A, was homologous to the NH2-terminal portion of alpha2-CB3. Thus, the order of the peptides in the alpha2 chain of this collagen is 1-O-4A-4B-2-3A-3B-5. Periodate oxidation and alkaline or acid hydrolysis of the CNBr fragments showed that all of the hydroxlysine-linked carbohydrate in the alpha2 chain was present in alpha2-CB4B. Carbohydrate analyses were most consistent with the existence of single monosaccharide and disaccharide units in this region.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Biosynthesis and regulation of type V collagen in diploid human fibroblasts

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.     

Structural studies on a glycoprotein isolated from alveoli of patients with alveolar proteinosis.

A major glycoprotein 36 000 molecular weight) has been isolated from lung lavage of patients with alveolar proteinosis and found to contain five residues of hydroxyproline, fifty residues of glycine, three residues of methionine, 3 mol of sialic acid, 4.4 mol of mannose, 4.0 mol of galactose, 6.0 mol of glucosamine, and 1 mol of fucose. Cyanogen bromide (CNBr) treatment of the glycoprotein resulted, as expected, in four peptides of apparent molecular weights of 18 000, 12 000, 5000 and 1000, respectively. The chemical compositions of the CNBr peptides indicate the presence of hydroxyproline and high amounts of glycine in all but one of the peptides; two of the four CNBr peptides contain carbohydrate. Gel filtration, acrylamide gel electrophoresis and end-group analyses of the native glycoprotein and its CNBr peptides indicate that the peptides are homogeneous. End-group analyses of the CNBr cleavage products assign the 18 000 molecular weight peptide to the NH2-terminal portion and the 1000 molecular weight peptide to the COOH-terminal portion of the native glycoprotein molecule. Pronase digestion of the 36 000 molecular weight glycoprotein, followed by gel filtration and cation exchange chromatography, resulted in two fractions. One fraction was acidic and contained all the carbohydrate, a high content of aspartic acid and no hydroxyproline. The other fraction was basic and contained 8.4% hydroxyproline, 14% proline, 28% glycine and no carbohydrate, suggesting the presence of collagen-like sequence in the peptide chain. Paper electrophoresis of the basic fraction demonstrated two components, the amino acid compositions of which are identical to those of collagen. Partial amino-terminal sequence analysis of one of the CNBr peptides (18 000 molecular weight) indicated the presence of -Fly-Pro-HyP-Gly-sequence in the peptide chain, which confirms our suggestion that collagen-like regions are present in the native glycoprotein molecule. Limited acid hydrolysis of the acidic fraction and subsequent fractionation of the acid hydrolysate using Dowex column yielded a fraction which produced brown colour with ninhydrin reagent. Paper chromatography of this fraction demonstrated a large component which also stained brown with ninhydrin reagent. After acid hydrolysis, this component was found to consist of equal amounts of asparitic acid and glucosamine, indicating that the N-acetylglucosamine of the oligosaccharides is linked to the asparagine residue of the peptide. No serine or threonine linkages are present.