Expression and characterization of recombinant protein S with the Ser 460 Pro mutation

To characterize the putative biochemical modifications induced by the Ser 4 60 to Pro (Heerlen) mutation in protein S (PS), we expressed both wild-type (wt) and mutated recombinant PS in HEK cells. In SDS-polyacrylamide gels, r-PS Heerlen migrated at 71 kDa whereas r-wt PS migrated at 73 kDa, a diffe rence abolished after deglycosylation by N-glycosidase, suggesting that the Ser 460 Pro mutation abolishes N-glycosylation of Asn 458. The affinity of r-wt PS and r-PS Heerlen for C4b-binding protein (C4b-BP) and for phosphol ipid vesicles was similar. Neither the enhancement of APC-dependent prolong ation of the APTT, nor the specific enhancement of FVa and FVIIIa proteolys is by APC in purified systems was affected by the mutation. However, the Se r 460 Pro mutation induced a slight conformational change in the SHBG domai n of the PS molecule, as shown by reduced binding affinity for monoclonal a ntibodies. The type III phenotype associated with the Heerlen mutation migh t thus result from a slightly modified rate of synthesis or catabolism. The resulting moderate decrease in the circulating PS concentration may modify the equilibrium between free PS and C4b-BP/PS complexes. (C) 2000 Elsevier Science Ltd. All rights reserved.