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Biomedical subjects

H Wiedemann

Publications and source records attributed to H Wiedemann.

At least 55 records · Page 3Linked to original sources
PubMed1984

Domain structure of cartilage proteoglycans revealed by rotary shadowing of intact and fragmented molecules.

The rotary-shadowing technique for molecular electron microscopy was used to study cartilage proteoglycan structure. The high resolution of the method allowed demonstration of two distinct globular domains as well as a more strand-like portion in the core protein of large aggregating proteoglycans. Studies of proteoglycan aggregates and fragments showed that the globular domains represent the part of the proteoglycans that binds to the hyaluronic acid, i.e. the hyaluronic acid-binding region juxtapositioned to the keratan sulphate-attachment region. The strand-like portion represents the chondroitin sulphate-attachment region. Low-Mr proteoglycans from cartilage could be seen as a globule connected to one or two side-chain filaments of chondroitin sulphate.

Animals↗
PubMed1984

Structure and interactions of heparan sulfate proteoglycans from a mouse tumor basement membrane.

Various forms of heparan sulfate proteoglycan were solubilized from the mouse Engelbreth-Holm-Swarm (EHS) sarcoma by extraction with 0.5 M NaCl, collagenase digestion and extraction with 4 M guanidine. They could be separated into high (greater than or equal to 1.65 g/ml) and low (1.38 g/ml) buoyant density variants. The high-density form from the NaCl extract and collagenase digest had Mr = 130000 and So20,W = 4.5 S and contained 4-10% protein, indicating Mr = 5 000-12 000 for the protein core. This proteoglycan exhibited polydispersity as shown by rotary shadowing electron microscopy and ultracentrifugation. An average molecule consisted of four heparan sulfate chains (Mr = 29 000) each with a length of 32 +/- 10 nm. The low-density form (Mr about 400 000) could not be completely purified and contained about 50% protein. As shown by radioimmunoassay, the various proteoglycans shared similar protein cores. Labeling of the tumor in vivo or in vitro demonstrated preferential incorporation of radioactive sulfate in the high-density form. The high-density proteoglycan interacted in affinity chromatography by virtue of its heparan sulfate chains with laminin, fibronectin, the globular domain NC1 and the triple helix of collagen IV. These interactions were abolished at moderate concentrations of NaCl (0.1-0.2 M) and in the presence of heparin, chondroitin sulfate or dextran sulfate. Interactions with the globule NC1 could also be demonstrated by velocity band centrifugation in sucrose gradients and a binding constant of about 10(6) M-1 was derived.

Animals↗
PubMed1984

Subunit structure and assembly of the globular domain of basement-membrane collagen type IV.

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.

Animals↗
PubMed1984

Isolation and characterization of a precursor form of M collagen from embryonic chicken cartilage.

A disulfide-cross-linked collagen has been extracted with neutral salt solutions from organ cultures of embryonic chick sternal cartilage. This collagen, which we term pM collagen, is presumed to be the native extracellular precursor molecule to disulfide-cross-linked collagen fragments recently described. Cleavage of pM collagen under native conditions with pepsin gives rise to the collagen fragments M1 and M2, which had also been isolated from pepsin extracts of chick hyaline cartilage [K. von der Mark, M. van Menxel & H. Wiedemann (1982) Eur. J. Biochem. 124, 57-62]. Native pM collagen was purified by DEAE-cellulose chromatography and agarose gel filtration. On agarose and following polyacrylamide gel electrophoresis, the unreduced molecule migrates with an apparent Mr of 300 000. Reduction of disulfide bridges produces two subunits with Mr 80 000 (pMa) and 60 000 (pMb) when compared with collagen standards. Cyanogen bromide cleavage of pMa and pMb, excised from dodecyl sulfate gels, resulted in different peptide maps, indicating that both components are genetically distinct polypeptide chains. The occasional appearance of the unreduced pM collagen as a doublet band on dodecyl sulfate gels and the observation that pMa and pMb occur in non-stoichiometric ratios suggests that pMa and pMb form separate native molecules, although their incorporation into a single pM molecule cannot be excluded. Native pM collagen was completely digested with bacterial collagenase, and contained hydroxyproline and proline in a ratio of 1.15:1, indicating the absence of significant non-collagenous domains. Thus it represents, despite several pepsinlabile sites, more likely a largely triplehelical, processed form of collagen rather than a procollagen-like molecule containing globular domains. Processing of pM collagen to M1 and M2 fragments or other intermediate forms was not observed in cartilage organ culture or in chondrocyte cell cultures within 18 h.

Animals↗
PubMed1984

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.

Animals↗
PubMed1983

The use of monoclonal antibodies to fragments of chicken type IV collagen in structural and localization studies.

In previous experiments, three pepsin-resistant fragments of type IV collagen were isolated from chicken gizzards and designated 7S, F3, and (F1)2F2 (Mayne, R., and Zettergren, J. G. (1980) Biochemistry 19, 4065-4072). In the present experiments, a series of monoclonal antibodies to type IV collagen were prepared, each one of which recognized an epitope present in only one of the three fragments. A high molecular weight fraction of type IV collagen (designated 7S + arms (215 nm)) was isolated after agarose gel filtration and characterized by electron microscopy after rotary shadowing and by gel electrophoresis. Analysis of 7S + arms (215 nm) by inhibition enzyme-linked immunosorbent assay demonstrated the presence of the epitopes for 7S and F3 but not for (F1)2F2. This result, therefore, provides additional evidence that the order of the pepsin-resistant fragments of chicken type IV collagen is 7S-F3-(F1)2F2.

Animals↗
PubMed1983

Electron-microscopical approach to a structural model of intima collagen.

Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.

Chemical Phenomena↗
PubMed1982

Characterization of a highly soluble collagenous molecule isolated from chicken hyaline cartilage.

Recently, we have isolated a new collagenous molecule from chicken hyaline cartilage after limited pepsin digestion. This molecule, which contains interchain disulfide bonds, has been called the high molecular weight fraction or HMW [Reese, C. A., & Mayne, R. (1981) Biochemistry 20, 5443-5448]. We now present a detailed model for the structure of HMW, the model being derived from analyses both of components of HMW obtained after denaturation either with or without reduction and from electron microscopic observation of replicas of HMW obtained after rotary shadowing. We propose that HMW is a typical, triple-helical collagen molecule of length 134 nm, in which one of the chains has been cleaved at a distance of 96 nm from one end of the triple helix, while the other two chains remain uncleaved. The result of this cleavage is the appearance of a recognizable kink in molecules of HMW when visualized after rotary shadowing.

Amino Acids↗
PubMed1982

Shape and assembly of type IV procollagen obtained from cell culture.

Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.

Animals↗
PubMed1982

Isolation and characterization of new collagens from chick cartilage.

Three unique collagen chains were isolated from chick sternal cartilage following pepsin solubilization of total cartilage collagens and removal of the predominant type II collagen by fractional salt precipitation. Native molecules containing 1 alpha, 2 alpha and 3 alpha chains precipitated between 0.7 M and 1.2 M NaCl at acidic pH and could be purified by chromatography on carboxymethyl-cellulose and agarose columns. Although similar to mammalian 1 alpha, 2 alpha and 3 alpha chains, differences in the mobilities on sodium dodecylsulfate gel electrophoresis, CNBr peptide profiles and amino acid composition were found. The 1 alpha and 2 alpha chains resemble, but are structurally distinct from, the chick alpha 1(V) and alpha 2(V) chains. The 3 alpha chain appears to be closely related to the alpha 1(II) chain, although some differences in the cyanogen bromide peptides suggest that they might be different gene products. In addition, two collagenous fragments of Mr 140 000 (M1) and 35 000 (M2) were found which precipitated at 2.0 m NaCl at acidic pH. Both fragments contain interchain disulfide bonds. The larger fragment was reducible to subunits of approximate Mr 120 000, 48 000, 28 000 and 11 000. The smaller fragment gave rise to peptides of Mr about 12 000 and 10 000 after reduction. By the technique of rotary shadowing the native, unreduced larger fragment M1 appeared as a slender rod-like molecule with a distinct bend approximately 40 nm from one end. We interpret this finding as indicative of a focal amino acid sequence irregularity, disrupting the triple-helical conformation.

Animals↗
PubMed1982

Structural and immunological characterization of type IV collagen isolated from chicken tissues.

Previously, a type IV collagen fraction was isolated from chicken gizzard and further fractionated into three components called F1, F2 and F3 [Mayne, R. and Zettergren, J.G. (1980) Biochemistry, 19, 4065-4072]. F1 and F2 were consistently isolated in a 2:1 proportion, and the existence of a small native fragment of structure (F1)2F2 was proposed. In the present series of experiments, a type IV collagen fraction was isolated from the chicken kidney and shown to consist almost entirely of F1 and F2 which were again present in a 2:1 proportion. Identical one-dimensional peptide maps for F1 and F2 from both sources were obtained by polyacrylamide gel electrophoresis of peptides obtained after cleavage with cyanogen bromide or Staphylococcus aureus V8 protease. The denaturation temperature of a preparation containing F1 and F2 in native form was determined by optical rotatory dispersion and a single melting curve was observed with a melting temperature of 33 degrees C. This result provides further supportive evidence that F1 and F2 exist as a native fragment (F1)2F2. Antibodies were prepared in rabbits against a type IV collagen fraction isolated from chicken gizzard, and immunofluorescent staining of a wide variety of basement membranes was demonstrated. Experiments were performed in which various type IV collagen fractions were observed in the electron microscope after rotary shadowing. The lengths of (F1)2F2 and F3 were 147 nm and 174 nm respectively, the sum of these lengths (321 nm) corresponding closely to the length of the major triple-helical domain of type IV collagen (326-328 nm). A specific cleavage site was located at a distance of 215 nm from the 7-S domain which, together with the length of (F1)2F2, gives a total length of 362 nm. This value corresponds closely to the maximum length of the arms which originate from the 7-S domain (355 nm) when type IV collagen was solubilized with a low concentration of pepsin. The results suggest that (a) type IV collagen isolated from the chicken gizzard is closely related, if not identical, to type IV collagen isolated from other tissues; (b) there is a single type IV collagen molecule of chain organization[alpha 1(IV)]2 alpha2(IV); (c) the order of the pepsin-resistant fragments within a type IV molecule is 7S-F3-(F1)2F2.

Animals↗
PubMed1981

A network model for the organization of type IV collagen molecules in basement membranes.

Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.

Animals↗
PubMed1978

Synthesis of collagen by chick calvaria in the presence of 2,2'-bipyridyl. Lack of the aminoterminal extension peptides.

A precursor of type I collagen was accumulated when cranial bones from embryonic chicken were cultured in vitro in the presence of 2,2'-bipyridyl. While biochemical studies demonstrate that the precursor was present as a covalently linked trimer, evaluation of electron micrographs of segment-long-spacing aggregates show that the molecules of this precursor have an extension at the carboxy terminus only. These data provide further evidence that the inter-chain disulfide bonds in procollagen are located at the carboxy-terminal extension, and secondly, that 2,2'-bipyridyl does not interfere with a normal cleavage of procollagen peptides at the amino terminus, but inhibits the cleavage of the carboxy-terminal procollagen peptides.

2,2'-Dipyridyl↗