Unmasking a hyaluronan-binding site of the BX7B type in the H3 heavy chainof the inter-alpha-inhibitor family

The inter-alpha -inhibitor (I alphaI) family gathers together several plasm a protease inhibitors such as I alphaI and pre-alpha -inhibitor (P alphaI) that are variously assembled from a set of polypeptide, chain precursors de signated H1P to H3P. In addition to their protease inhibitory activity, a m ajor physiological function of I alphaI family members is hyaluronan (HA) b inding and HA-dependent stabilization of the extracellular matrix surroundi ng various cell types. Also, binding of HA to these molecules has been show n to be an important event in tumor cell proliferation and rheumatoid arthr itis. However, how HA and I alphaI family members first recognize each othe r has so far remained elusive. The so-called BX7B domain found in some HA-b inding proteins is an HA-binding site in which B represents a basic amino-a cid residue and X represents any nonacidic residue. This domain has now bee n identified in the N-terminal end of H3P that is a precursor of P alphaI. A series of wild-type or mutant recombinant H3P chains produced with a mous e cDNA expressed in Escherichia coli allowed us to demonstrate that this do main binds HA in a noncovalent fashion. Furthermore, unmasking this HA-bind ing activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purifi ed from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. The refore, in vivo, the HA-binding activity of the mature H3 chain within P al phaI may vary with the folding and/or fragmentation of this protein.