Biosynthesis of chick type VI collagen. I. Intracellular assembly and molecular structure

A monospecific rabbit antiserum to pepsin-extracted chick gizzard type VI collagen was used to characterize the intact forms of type VI collagen in tissues and cultured cells. Immunoblotting of gizzard extracts revealed polypeptides of Mr ranging from 260,000 to 140,000. Components of about Mr = 260,000, 150,000, and 140,000, each with a different peptide profile, were immunoprecipitated from labeled matrix-free chick embryo cells. Cleavage of the immunoprecipitated polypeptides with pepsin generated pepsin-resistant fragments of about Mr = 70,000, 55,000, and 45,000 that represent the alpha 1(VI), alpha 2(VI), and alpha 3 (VI) fragments. Immunoblotting with affinity-purified antibodies indicated that the Mr = 150,000 is the intact parent polypeptide of the alpha 1(VI) pepsin; the Mr = 140,000 of the alpha 2(VI) pepsin, and the Mr = 260,000 of the alpha 3(VI) pepsin. Association of the three parent chains was studied by pulse-chase experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under nonreduced conditions. A complex of Mr = 500,000 is already present intracellularly at the end of a 7-min pulse and increases considerably with time while the three unassembled chains show a comparable decrease. After 5-15 min of chase larger forms appeared along with small amounts of aggregated material that did not enter the gel. Analysis of the immunoprecipitate by diagonal electrophoresis indicated that the component of Mr = 500,000 and the larger forms dissociated into the Mr = 260,000, 150,000, and 140,000 polypeptides. Sedimentation profile of a labeled cell extract on a 5-20% sucrose gradient under nondenaturing conditions confirmed the presence of three different peptides in the complex.     

Biosynthesis of chick type VI collagen. II. Processing and secretion in fibroblasts and smooth muscle cells

The biosynthesis of type VI collagen was studied in "matrix-free" chick embryo smooth muscle cells and fibroblasts. Omission of ascorbate from the culture affected to a great extent the secretion in fibroblasts but had a very minor effect on smooth muscle cells. Quantitative analysis of the secretion process in continuous time course and in pulse-chase experiments confirmed that fibroblasts and smooth muscle cells secreted type VI collagen with the same chain composition but with different kinetics: after 4 h of chase more than 60% of the labeled type VI collagen was present in the culture medium of fibroblasts, whereas at the same time interval less than 25% was secreted by smooth muscle cells. The different kinetics depends on intrinsic properties of the cells, since it was detected also in adherent cells. However, even in fibroblasts, secretion of type VI collagen was much slower than secretion of fibronectin, of which more than 50% was already in the cell medium after 1 h of chase. Treatment of the cells with inhibitors of hydroxylation and glycosylation caused a shift in mobility that revealed a size heterogeneity in the Mr = 260,000 subunit. No evidence of processing was observed in chick cells for any of the subunits that were synthesized and secreted uncleaved. In addition, after several days of chase the Mr of the subunits of type VI collagen isolated from the matrix remained unchanged, thus excluding that in the chick even a partial or incomplete processing takes place.     

Characterization of a cDNA encoding subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum.

The primary structure of subunit VI of cytochrome c oxidase from the slime mold Dictyostelium discoideum has been determined by sequencing cDNA and N-terminus of the protein. The 92 amino acid residues long polypeptide (Mr = 10,535) shows homology with subunit IV of mammalian and subunit V of yeast cytochrome c oxidase. Though smaller and synthesized without a cleavable presequence, the slime mold oxidase subunit maintains the presence of a putative membrane spanning region.     

The exon organization of the triple-helical coding regions of the human alpha 1(VI) and alpha 2(VI) collagen genes is highly similar.

The alpha 1(VI) and alpha 2(VI) chains, two of the three constituent chains of type VI collagen, are highly similar in size and domain structure. They are encoded by single-copy genes residing in close proximity on human chromosome 21. To study the evolution of the type VI collagen genes, we have isolated and characterized genomic clones coding for the triple-helical domains of the human alpha 1(VI) and alpha 2(VI) chains, which consist of 336 and 335 amino acid residues, respectively. Nucleotide sequencing indicates that, in both genes, the exons are multiples of 9 bp in length (including 27, 36, 45, 54, 63, and 90 bp) except for those encoding for regions with triple-helical interruptions. In addition, the introns are positioned between complete codons. The most predominant exon size is 63 bp, instead of 54 bp as seen in the fibrillar collagen genes. Of particular interest is the finding that the exon structures of the alpha 1(VI) and alpha 2(VI) genes are almost identical. A significant deviation is that a segment of 30 amino acid residues is encoded by two exons of 54 and 36 bp in the alpha 1(VI) gene, but by a single exon of 90 bp in the alpha 2(VI) gene. The exon arrangement therefore provides further evidence that the two genes have evolved from tandem gene duplication. Furthermore, comparison with the previously reported gene structure of the chick alpha 2(VI) chain indicates that the exon structure for the triple-helical domain of the alpha 2(VI) collagen is strictly conserved between human and chicken.     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Characterization of the precursor form of type VI collagen

Well characterized monospecific antisera against pepsin-extracted bovine type VI collagen were used to identify and characterize the intact form of type VI collagen. In immunoblotting experiments the antisera reacted with the pepsin-resistant fragments of the alpha 1(VI) and alpha 3(VI) chains, but not with the fragment of the alpha 2(VI) chain. Extracts obtained from uterus and aorta with 6 M guanidine HCl contained two immunoreactive polypeptides of Mr = 190,000 and 180,000 based on globular protein standards. Cleavage of extracts with pepsin generated the previously characterized pepsin-resistant fragments of alpha 1(VI) and alpha 3(VI), indicating that the higher molecular weight polypeptides represent the intact parent chains, alpha 1(VI) and alpha 3(VI). Digestion of extracts with bacterial collagenase released an Mr = 100,000 noncollagenous fragment from the alpha 1(VI) chain. Thus, intact type VI collagen in tissues contains a relatively short triple helical domain and at least one very large globular domain which is sensitive to pepsin but resistant to collagenase digestion. Immunoblotting revealed a polypeptide of Mr = 240,000, which we suggest represents the pro-alpha 1(VI) chain, in the culture medium of bovine fibroblasts. Bands intermediate in molecular weight between 240,000 and 190,000 were identified in cell layers. These findings establish type VI collagen as a protein with very large nontriple helical domains, a property that undoubtedly plays an important role in its function.     

Nuclear genes for cytochrome c oxidase subunits of Neurospora crassa. Isolation and characterization of cDNA clones for subunits IV, V, VI, and possibly VII

We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N-terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established.     

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.     

Entamoeba histolytica: cDNAs cloned as 30kDa collagen-binding proteins (CBP) belong to an antioxidant molecule family. Protection of hamsters from amoebic liver abscess by immunization with recombinant CBP

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30 kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69 bp deletion at the 5' end that is present in C13 and encodes for a Glu-Cys-Lys rich region and a four amino acids repeat (Glu-Lys-Glu-Cys). Purified fusion proteins from these cDNA clones were able to bind native type I collagen gels in a pH, calcium, ionic strength, and temperature dependent way. The binding of pgtC13 to collagen gel was time and temperature stable, while pgtC7 binding was not, suggesting that the deleted region in C7 is important for the binding. The clones reported here partially encode a 30 kDa CBP that also belong to an antioxidant molecule family. We demonstrated that the fusion protein pgtC13 is immunogenic and partially protective as a subunit vaccine in the hamster model of amoebic liver abscess.     

Characterization of porcine plasma fibronectin and its fragmentation by porcine liver cathepsin B

Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Structural and functional relationships between h- and l-caldesmons.

Two different Mr forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified. h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells. Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511; Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W-G. (1989) J. Biol. Chem. 264, 13873-13879). The calculated Mr of this protein from its primary structure is 88,743. Here, the nucleotide and deduced amino acid sequences of l-caldesmon have been determined by cloning and sequencing the cDNA from chick brain and compared with those of h-caldesmon. The l-caldesmon cDNA encodes a sequence of 517 amino acids with the calculated Mr of 58,844. Two isoforms of caldesmon conserve the completely identical sequences in the NH2- and COOH-terminal domains except for the insertion of Ala-508 in l-caldesmon. Interestingly, the central repeating sequence of h-caldesmon (residues 201-447) is deleted in the l-caldesmon molecule. The short NH2-terminals of two caldesmons individually show the unique sequences. The results of Northern and Southern blot analyses suggest that two mRNAs (4.8 and 4.1 kilo-bases) coding for caldesmon isoforms may be generated from a single gene by alternative splicing. Using a series of truncated caldesmons expressed in Escherichia coli, the common calmodulin-, tropomyosin-, and actin-binding sites and the minimum regulatory domains, which are involved in the Ca2(+)-dependent regulation of actin-myosin interaction, have been identified within the limited consensus sequences (residues 381-433 for l-caldesmon and residues 636-688 for h-caldesmon).     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mapping technique for probing the structure of proteoglycan core molecules

Our previous work showed that treatment of chick embryo cartilage proteoglycan (PG-H) with chondroitinase-AC II and keratanase yielded a protein-rich core fraction having enzymatically modified linkage oligosaccharides. The core sample has now been analyzed by tryptic peptide mapping, in which the isolated core sample contained in a single Coomassie blue-staining band from a dried slab gel is radioiodinated and treated with trypsin, and the resultant tryptic peptides are displayed two-dimensionally on a silica gel thin layer plate. The map thus obtained exhibited 22 major peptide spots, the resolution and location of which were reproducible. In order to identify regions of the core polypeptide from which the tryptic peptides are derived, PG-H was cleaved with clostripain under conditions that yield a hyaluronic acid-binding fragment with an apparent Mr = 150,000 and chondroitin sulfate-peptide clusters of smaller molecular sizes. Although the peptide maps of the two size classes of clostripain fragments differed significantly from each other, the patterns of spots, as a whole, were extensively similar to those observed with the intact core molecule. These results have provided additional evidence that PG-H has a single, nonvariable core protein structure. In addition, the technique used here will provide a versatile method for the identification of genetic types in this increasingly complex family of matrix macromolecules.     

Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.