Arnold U, Schierhorn A, Ulbrich-Hofmann R
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
J Protein Chem. 1998 Jul;17(5):397-405. doi: 10.1023/a:1022562316513.
The influence of glycosylation on proteolytic degradation was studied by comparing cleavage sites in ribonuclease A (RNase A) and ribonuclease 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 desorption/ionization mass spectrometry and compared with those in RNase A [Arnold et al. (1996), Eur. J. Biochem. 237, 862-869]. RNase B was cleaved by subtilisin even at 25 degrees C at Ala2-Ser21 as known for RNase A. Under thermal unfolding, the peptide bonds Asn34-Leu35 and Thr45-Phe46 were identified as primary cleavage sites for thermolysin and Lys31-Ser32 for trypsin. These sites are widely identical with those in RNase A. Treatment of reduced and carbamidomethylated RNase A and RNase B with trypsin led to a fast degradation and revealed new primary cleavage sites. Therefore, the state of unfolding seems to determine the sequence of degradation steps more than steric hindrance by the carbohydrate moiety does.
