CONVERSION OF THE KUNITZ-TYPE MODULE OF COLLAGEN-VI INTO A HIGHLY-ACTIVE TRYPSIN-INHIBITOR BY SITE-DIRECTED MUTAGENESIS

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen alpha 3(VI) chain was previously shown to lack inhibitory act ivity for proteases with trypsin-like specificity and some other prote ases, We have now prepared mutants in the binding loop region includin g the P1' site (D2889-->A), the P2' site (F2890-->R) and the P3 site ( T2886-->P) and in a more remote region (W2907-->V) either as individua l substitutions or combinations of them. These mutants were analyzed f or their kinetics of binding to trypsin by surface plasmon resonance a nd for their capacity to inhibit various proteases. Single substitutio ns (D-->A, T-->P, W-->V) showed an effect only for D-->A which bound t o trypsin with K-d = 0.25 mu M A 25-100-fold increase in affinity was observed for the double mutants T-->P/D-->A and F-->R/D-->A and approa ched the affinity of aprotinin (K-d approximate to 0.01 nM) in two dif ferent triple mutants. These affinities correlated well with the inhib itory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely i dentical improvement in inhibitory capacity was also observed for leuc ocyte elastase but not for thrombin. These data could be interpreted i n terms of steric interferences or lack of hydrogen bonding of a few c ritical residues based on three-dimensional structures available for t he C5 domain.