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 NCI were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously described 7-S collagen domain. The pepsin-solubilized form lacked domain NCI 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 NCI domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NCI) was estimated to be about 800 nm comprising the length of two molecules.Type IV collagen is a unique member among the collagenous proteins and is considered to be the major structural component of basement membranes [I 1. Biosynthetic studies have shown that the constituent chains of type IV collagen ( M , about 180000) are larger than those of interstitial collagens and procollagens, and that they are not substantially processed when deposited in the matrix [2-51. A further unique feature are frequent interruptions of the triple helix as indicated by sequence analysis [6]. This explains the protease sensitivity of native type IV collagen [7,8] and possibly causes a greater flexibility of the large triple-helical segments. These molecules also possess another short triple-helical segment (7-S domain) which appears to be part of a rather compact fragment named 7-S collagen [9,10].Electron microscopical studies have demonstrated that basement membranes have a rather amorphous appearance [I I], quite different from the cross-striated fibrillar structures of collagenous proteins observed in interstitial connective tissue. X-ray diffraction studies of stretched lens capsules have indeed indicated a poorly ordered fibrillar array [12]. On the basis of these observation Kefalides [I] suggested a model for the basement membrane matrix which envisions sheets of type IV collagen to be cross-linked to alternating layers of non-collagenous proteins. Ot...
Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd‐I (positions 1–905) comprising the N‐terminal globule and rod‐like domain and Nd‐II corresponding mainly to the C‐terminal globule (position 906–1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N‐terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N‐terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31–56 kDa) connected by either a rod‐like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd‐I binding to collagen IV and fragment Nd‐II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd‐I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.
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.
The EHS sarcoma (named after its discoverer, Engelbreth-Holm, and its primary characterizer, R. Swarm) contains an extracellular matrix of basement membrane. The collagenous component of the membrane was extracted with dilute acetic acid from tumors grown in lathyritic mice and separated from other proteins by chromatographic methods. This collagen has a composition resembling that reported for other basement membrane collagens. The polypeptide chains of the tumor collagen are linked by disulfide bonds and migrate after reduction intermediate between the CI chains und components of type-I collagen. The circular dichroism spectra obtained from it resemble those obtained from other collagens.The intact protein contained about 280 residues of glycine per 1000 residues. After reduction of disulfide bonds in the basement membrane collagen, treatment with either pepsin or trypsin gave rise to resistant fragments containing one-third glycine. These fragments formed segment-long-spacing crystallites 210 nm long in the case of those treated with pepsin and 350 nm long in the case of those treated with trypsin. The band pattern differs from that found with other collagens.Antibodies prepared against the tumor collagen were found to localize to the tumor matrix and to known basement membranes occurring in mouse tissues. The data suggest that these basement membranes may contain a collagenous protein similar to the tumor protein.
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