Abstract
Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.
Highlights
Collagenbinding on Caz+uptake by Matrix vesicles (MV) isolatedfrom chicken growth plate cartilage were treated with pure bacterial collagenaseand 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens
MV, chorin CII), one of three annexins found in suggesting that annexin V is involved in mediating the binding of type I1 and X collagens to the MV surface
The resultsof our study indicate that interaction withI1type and X collagens, which are themajor collagenous components in hypertrophic cartilage, regulates uptake and accumulation of Ca2+by MV
Summary
MV were isolated from growth plate cartilage of W-week-old broiler strain chickens by enzymatic tissue digestion as described previously [22]. Chymotrypsin lFeatment of Collagenase,1M NaC1-treatedMV-After described previously[27].After blockingwith a solution of low fat milk protein, blotted proteins were immunostained with rabbit anti-annexin collagenase and 1 M NaCl treatment, aliquots of MV pellets were further V IgG or rabbit anti-human type X collagen IgGfraction, using peroxiincubated in 50%Ca", SCL containing 1%chymotrypsin (Type I1 from dase-conjugated goat anti-rabbit IgG (Sigma) in a 1:lOOO dilution as bovine pancreas, Sigma) for 2 h at 37 "C. second antibody and a-chloronaphthol as a color substrate. NaCl or collagenase, 1M NaCl, chymotrypsin-treated MV pellets were incubated with different concentrations (50, 20, 10, 5, 2 and 1 pg) of purified native or pepsin-treated or denatured type I1 collagen or purified native or pepsin-treated type X collagen in 50% Ca2'-containing SCL for 18 h a t 4 "C. The samples were analyzed on a Beckman amino acid analyzer (model 6300) using ninhydrin reagent
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