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Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

The N-terminal non­collagenous domain NCll of the human collagen α1(XVI) chain was obtained as a recombinant 35-kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic trimming at a basic cleavage motif indicating a similar release in vivo. Domain NCll showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NCll and were used for the analysis of other cell clones transfected with the full-length α(XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide-bonded 200-kDa polypeptides possessing NCll epitopes. This material was partially resistant to pepsin, indicating the formation of αl(XVI) chain homotrimers with a triple-helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35–150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple-helix formation. Edman degradation demonstrated the origin of some from the N-terminal and of one from a more C-terminal position of collagen XVI. This extensive degradation may be explained by the release of NCll and by further cleavages within some of the nine interruptions of the triple-helical domain of the α1(XVI) chain. Whether this process also occurs in situ remains to be shown. [ABSTRACT FROM AUTHOR]

The Kunitz-type inhibator motif (domain C5) present at C-terminus of the human collagen α3(VI) chain was prepared in a recombinant from the culture medium of stably transfected protease treatment. The recombinant protein was disulfide bonded and showed a high sability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikerein and several other protease, which could be due to a few unusual substitutions of C5 epitopes in normal human serum and fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several α3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicates the rapid loss of the C5 domain after biosynthesis of collagen VI. [ABSTRACT FROM AUTHOR]

Full-length alpha 1(VI) and α 2(VI) cDNAs in an eukaryotic expression vector were used to obtain stably transfected human kidney cell clones and to purify these collagen-VI chains in substantial quantities from the culture medium. Both chains appeared mainly as monomers together with some dimers that were disulfide linked through their C-terminal globular domains. Despite sufficient hydroxylation of proline and lysine residues, the chains did not form a triple-helix, as shown by electronmicroscopy, CD spectra and pepsin sensitivity. Digestion of the chains with bacterial collagenase released the N-terminal and C-terminal globular domains, which were identified by their size and partial sequences. They showed a substantial content of alpha-helical conformation and a distinct globular structure after rotary shadowing. Antibodies could be raised that distinguished between the two chains and reacted with the globular domains. The α 2(VI) but not the α 1(VI) chain showed binding to a heparan sulfate proteoglycan (perlecan), fibronectin and pepsin-solubilized collagen VI. Purified globular domains did not bind these ligands indicating the localization of binding sites within the triple-helical domain. Both chains showed a distinct affinity for heparin but failed to bind to various collagen types. [ABSTRACT FROM AUTHOR]

Temporal and spatial expression of α1 (IV), α2 (IV), α3 (IV), α4 (IV), α5 (IV), and α6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of β1, β4, α1, α2, α3, α5, α6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of α2 (IV) and α6 (IV) chains was estimated by northern blots. The earliest expression of α1 (IV) and α2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, α1 (IV) and α2 (IV) chains were noted in basal keratinocytes and as a broad band (10 μm) in the adjacent dermis. At this stage 80% of the α2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the α1 (IV) and α2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dennis. The α6 (LV) collagen chains were expressed much later at 36-d cultures and the α5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. α6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and β1 integrin subunits in 14-.d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-β1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3 [IV]) that contains the collagen IV ligand for α1β1, α2β1 integrins, altered the pattern of collagen IV deposition. [ABSTRACT FROM AUTHOR]