INFLUENCE OF THE CARBOHYDRATE MOIETY ON THE PROTEOLYTIC CLEAVAGE SITES IN RIBONUCLEASE-B

The influence of glycosylation on proteolytic degradation was studied by comparing cleavage sites in ribonuclease A (RNase A) and ribonuclea se B (RNase B), which only differ by a carbohydrate chain attached to Asn34 in RNase B. Primary cleavage sites in RNase B were determined by identifying complementary fragments using matrix-assisted laser desor ption/ionization mass spectrometry and compared with those in RNase A [Arnold et al. (1996), fur. J. Biochem. 237, 862-869]. RNase B was cle aved by subtilisin even at 25 degrees C at Ala20-Ser21 as known for RN ase A. Under thermal unfolding, the peptide bonds Asn34-Leu35 and Thr4 5-Phe46 were identified as primary cleavage sites for thermolysin and Lys31-Ser32 for trypsin. These sites are widely identical with those i n RNase A. Treatment of reduced and carbaminomethylated RNase A and RN ase B with trypsin led to a fast degradation and revealed new primary cleavage sites. Therefore, the slate of unfolding seems to determine t he sequence of degradation steps more than steric hindrance by the car bohydrate moiety does.