Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

Abstract

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.

Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc

“…The antibodies used were MAb CS-56 to native CS a, b anti-aggrecan G1 domain c anti-aggrecan G3 domain d, Mab 12C5 to versican G1 domain e and Mab 5D5 to versican G3 domain f aggrecan acting as lubricating agents is a new role for these proteoglycans in the IVD but consistent with roles proposed for CS-PGs in tendon. Furthermore, type VI collagen is capable of interacting with hyaluronan, a well known lubricating component of synovial fluids [28,29,31,44,52,53] and this association may also facilitate the lubrication of collagen bundles.…”

Extracellular matrix in the trabecular meshwork

“…(Hessle and Engvall 1984;Bruns et al 1986;Engvall et al 1986;Knupp et al 2006) The type VI collagen N-terminal repeats bind hyaluronan and heparin, while the C-terminal domains are involved in self-assembly. (Kielty et al 1992b;Specks et al 1992;Ball et al 2001;Ball et al 2003) Decorin and biglycan associate with the N-terminal end of the central collagenous domain, affecting fibril formation. This domain also contains RGD sequences, which are responsible for cell-association.…”

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA

“…Expression of PR3 mRNA of the expected size (0.9 kb) in HMC-1/PR3 cells was detected by Northern blot analysis (performed as described elsewhere [25]), but not in HMC-1/VEC or nontransfected HMC-1 parent cells (not shown).…”