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 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.
Arnold
U
U
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Schierhorn
A
A
Ulbrich-Hofmann
R
R
D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D006868:Hydrolysis; D011487:Protein Conformation; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002241:Carbohydrates; D012260:Ribonucleases; C029600:ribonuclease B; D013381:Subtilisins; D014357:Trypsin; D013820:Thermolysin