A large portion of the N‐terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1–1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9‐N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease‐sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9‐N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9‐N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9‐N2 for the triple helical region of collagen VI suggesting a role of the N‐terminal globule in the self‐assembly of microfibrils. No or only little binding was, however, observed to fibril‐forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9‐N2 was also an inactive substrate for cell adhesion.
A monoclonal antibody generated against the isolated extracellular matrix (ECM) of the medusa Podocoryne carnea M. Sars (Coelenterata, Cnidaria, Hydrozoa) stains a fibrillar component of the Podocoryne ECMs in immunohistochemical preparations. The antigen shows a different staining pattern according to the type of ECMs from the animals life cycle. In ontogeny the epitope first appears after gastrulation in the planula larva as single widely dispersed small fibrils, which later accumulate to form a dense meshwork in the larval ECM. The distribution of the antigen strongly suggests an important role of the molecule to cover the biomechanical needs of the animal. In immunoblots one band with a size of 330 kDa is detectable in the polyp ECM, whereas in the outer ECM of the medusa a 340-kDa band is observed. Both the 330- and the 340-kDa bands appear when probed on the inner ECM of the medusa or on ECMs of the larva. The antibody was used to isolate a cDNA clone from an expression library. The deduced amino acid sequence of this cDNA fragment reveals a molecular structure composed of tandemly repeated epidermal growth factor-like repeats interrupted by a second cystein-rich motif first found in the latent transforming growth factor beta binding protein. Comparison of the sequence to the data bases indicates < 40% identity to human fibrillins. The presence of fibrillin-like beaded microfibrils in the ECM of P. carnea is furthermore demonstrated by electron microscopy after rotary shadowing. Our results demonstrate for the first time the existence of this noncollagenous interstitial ECM protein in invertebrates and suggest that the structure and the function of fibrillin have been conserved during evolution.
The C-terminal non-collagenous domain of the surfactant glycoprotein SP-A was shown to be essential for its correct folding and assembly, as judged by the secretion of various deletion mutants transiently expressed in COS cells. A deletion mutant coding for this domain was successfully secreted while the expression of the collagenous domain only did not lead to any detectable secretion. Deletion mutants lacking small parts of the non-collagenous domain interfered more or less with the correct folding and assembly of the molecule, thus either reducing or inhibiting the secretion. These data suggest that three prefolded non-collagenous domains register and act as a nucleation center for the folding of the collagenous triple helix which proceeds in a zipper-like fashion towards the N-terminus.The glycoprotein SP-A was first identified to be associated with surfactant lipids by King and Clements [I]. It was shown to be the most abundant of the surfactant proteins [2,3]. SP-A was localized in lamellar bodies [4, 51 and tubular myelin [6, Besides its cooperative effect on the phospholipid spreading in the presence of hydrophobic surfactant proteins SP-B and SP-C [8], it is mostly involved in metabolic aspects of the surfactant system. SP-A has been described to enhance the uptake of phospholipids by type I1 pneumocytes [9] and to inhibit the phospholipid secretion of the same cells [lo-121. In this context the protein was shown to bind specifically to the type I1 pneumocytes [13, 141. SP-A, which may be a calciumdependent lectin [15, 161, was recently shown to be involved in the activation of the defence system of the lung [17].The primary structure of SP-A [18, 191 indicated the presence of two different structural domains, a collagenous triple helix and a globular domain located at the C-terminus. These domains assemble to a complex hexameric structure resembling a flower bouquet which is composed of 18 polypeptide chains [20, 211. First studies on the dissociation and refolding of SP-A suggested the lollypop-shaped monomers, composed of three a-chains, to be the first structural element formed WI.The C-terminal globular domain may, in analogy to the collagens [23], function as a nucleation site for triple helix formation. To address this question, we introduced various deletions in the collagenous and the non-collagenous domains of human SP-A and monitored for the correct folding, via their secretion, using a transient mammalian expression system. 71.
: we have reported a dumbbell-shaped structure of dystrophin molecule (Murayama et al., 1990;Sato et al., 1992). However its small population has raised a possibility that the dumbbell structure was due to a contaminant protein, such as type VI collagen of aorta origin. In the present study, type VI collagen was purified from bacterial collagenase-treated aorta of the rabbit. Its rotary shadowed images were very similar to those present in the dystrophin preparations.However, the antiserum to type VI collagen raised in a mouse did not react with dystrophin samples. A tentative explanation for the small population of the dumbbell structure is described.Key words : Dystrophin; type VI collagen; rabbit skeletal muscle.The molecular shape of dystrophin is important in the understanding of the physiological role of dystrophin that binds to the glycoprotein complex of the muscle cell membrane and to the cytoskeletal structure including actin filaments. 1),2)There have been two reports as to the molecular shape of dystrophin: 175 nm flexible rod from chicken gizzard3) and 130 nm dumbbell-shaped tetramer from rabbit skeletal muscle.4) The problem in the latter was its small population in the purified dystrophin preparations.5) There could be a contaminant protein with the particular structure. One of such candidates is type VI collagen of aorta origin.6)'7) Therefore, in the present study, type VI collagen was purified from bacterial collagenase-treated aorta of the rabbit. Immunoblot tests with the antiserum to the type VI collagen showed that contamination with the collagen was negligible in the dystrophin preparations.Dystrophin was prepared from rabbit skeletal muscle as described5) (Fig. 1A). Type VI collagen was extracted from bacterial collagenase-treated aorta of the rabbit. The al and a2 subunits (140 kDa) of type VI collagen were isolated by DEAE-Sephadex and hydroxylapatite column chromatographyg) (Fig. 1B). Two mice were immunized by injecting the 140 kDa bands cut out from SDS electrophoresed gels (Fig. 1B1). The dumbbell structures of dystrophin (Fig. 2a, c, d, e, f and g) were strikingly similar to those of type VI collagen (Fig. 2b, h, i). The size (130 nm) of the tetramers was very similar to each other, as reported with dystrophin5) and type VI collagen.7) Long chains were seen frequently observed with type VI collagen (Fig. 2b, i), whereas short chains were seen in dystrophin preparations (Fig. 2a, c, d, e, f and g).Monoclonal antibodies, A1C and 4-4C5, to dystrophin did not react with type VI collagen (Fig. 3B2, 3) and likewise the antiserum to type VI collagen did not react with dystrophin (Fig. 3A4). Using the antiserum to type VI collagen, the possibility of type VI collagen contamination with various dystrophin preparations was tested. The antiserum detected the 140 kDa subunits of type VI collagen (4 ng/lane, 4 mm wide). As described in a previous paper,5) the dumbbell structure was abundantly present in the first fraction, where dystrophin began to be eluted in HPLC gel column chrom...
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