Isolation from bovine elastic tissues of collagen type VI and characterization of its form in vivo

Abstract

Foetal-bovine nuchal ligament and aorta, together with adult-bovine aorta and pregnant uterus, were extracted under dissociative conditions in the absence and in the presence of a reducing agent. A collagenous glycoprotein of Mr 140000 [designated component 140K(VI)], identified in these extracts as the major periodate/Schiff-positive component, was shown to be related to collagen type VI. Digestion of non-reduced extracts with pepsin yielded periodate/Schiff-positive peptides that, on the basis of their electrophoretic mobilities, amino acid analyses and peptide 'maps', were identical with type VI collagen fragments prepared by standard procedures. It is concluded that collagen type VI occurs in vivo as molecule comprising three chains of Mr 140000 in which the helical domains account for about one-third of each polypeptide. Biosynthetic experiments with nuchal-ligament fibroblasts in culture demonstrated that a bacterial-collagenase-sensitive [3H]fucose-labelled glycoprotein, Mr 140000, was immunoprecipitated from culture medium by a specific antibody to the pepsin-derived form of collagen type VI. This result suggests that the collagenous polypeptides [140K(VI) components] represent the biosynthetic precursors of type VI collagen that do not undergo processing to smaller species before deposition in the extracellular matrix. Analyses of 5M-guanidinium chloride extracts of tissues with markedly different elastin contents and at different stages of development suggested that there was no relationship between collagen type VI and elastic-fibre microfibrils, a conclusion supported by the observation that the immunoprecipitated glycoprotein, Mr 140000, was distinct from the glycoprotein MFPI, Mr 150000, believed to be a constituent of these microfibrils [Sear, Grant & Jackson (1981) Biochem. J. 194, 587-598].

Type VI collagen and glycoprotein MFPI are distinct components of the extracellular matrix

“…The possible relationship between the secreted glycoprotein of Mr 140000 and 150000 and the 140K(VI) component of type VI collagen [12] was investigated by using a specific anti-(type VI collagen) antibody. Comparable experiments with anti-(microfibrillar protein) serum could not be carried out as this antiserum [3] is no longer available, and attempts in this and in other laboratories to produce an antiserum with similar specificity have been unsuccessful.…”

“…for three determinations. Peptide ' mapping' was carried out on [3H]prolineand [3H]fucoselabelled components after digestion with Staphylococcus aureus V8 proteinase or CNBr [12].…”

“…More-recent studies have described the extraction and purification of another collagenous glycoprotein (Mr 140000) from a number of tissues varying markedly in their elastin contents [10][11][12][13]. This glycoprotein represents a reduced subunit of the intact form of type VI collagen and is designated 140K(VI) component [12]. Type VI collagen is not, however, aconstituent ofelastin-associated microfibrils [12,14,15].…”

“…This glycoprotein represents a reduced subunit of the intact form of type VI collagen and is designated 140K(VI) component [12]. Type VI collagen is not, however, aconstituent ofelastin-associated microfibrils [12,14,15]. The obvious similarity between these newly synthesized and tissue-extracted glycoproteins led, not surprisingly, to proposals that they were probably identical [10, 14, [16][17][18].…”

Type VI collagen is associated with microfibrils and oxytalan fibers in the extracellular matrix of periodontium, mesenterium and periosteum

“…However, this is less clear for type VI collagen. Several authors have extracted microfibrillar components of various soft connective tissues and analysed this material biochemically and microscopically ( 5 , 13,14). Although these studies demonstrated that the extracted microfibrillar structures contained or even consisted of type VI collagen, it was unclear whether the microfibrils originated from the oxytalan system.…”