PROTEINS OF THE INTER-ALPHA-TRYPSIN INHIBITOR FAMILY STABILIZE THE CUMULUS EXTRACELLULAR-MATRIX THROUGH THEIR DIRECT BINDING WITH HYALURONIC-ACID

We have previously identified a glycoprotein of the inter-alpha-trypsi n inhibitor family of proteins as a serum factor responsible for the s tabilization of the expanding cumulus mass. In this study, the mechani sm of interaction of this cumulus extracellular matrix stabilizing fac tor (cESF) with hyaluronic acid (HA) has been explored. It was found t hat the pH optimum for binding of cESF and HA is 7 and that binding is sensitive to ionic strength. The dissociation constant is about 1.9 x 10(-8) ar in 10 mM sodium phosphate buffer (pH 7.2). Circular dichroi sm studies show that cESF contains about 24% alpha-helical and 42% bet a-sheet structure. Gross conformational changes in cESF, however, are not detected in the presence of HA. me also found that modification of lysine residues of cESF with citraconic anhydride greatly reduced its binding with HA and completely abolished its cumulus stabilizing acti vity, and deblocking lysine residues restored its capacity to bind wit h HA and its cumulus matrix stabilizing activity. This evidence suppor ts the hypotheses that cESF stabilizes the expanding cumulus extracell ular matrix by directly binding with HA and that cESF may serve as a s tructural protein to organize the formation of the cumulus extracellul ar matrix. Our evidence also supports the view that binding of cESF an d HA is through a stereo-specific charge interaction. Putative binding sites of cESF that interact with HA are postulated.