The glycosylation of the aspartic proteinases from barley (Hordeum vulgare L.) and cardoon (Cynara cardunculus L.).
Plant aspartic proteinases characterised at the molecular level contain one or more consensus N-glycosylation sites [Runeberg-Roos, P., Tŏrmäkangas, K. & Ostman, A. (1991) Eur. J. Biochem. 202, 1021-1027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem, 232, 77-83; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 762-768]. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) aspartic proteinase (Asn333) and the cardoon (Cynara cardunculus L.) aspartic proteinase, cardosin A (Asn70 and Asn363). The oligosaccharides from each site were released from peptide pools by enzymatic hydrolysis with peptide-N-glycanase A or by hydrazinolysis and their structures were determined by exoglycosidase sequencing combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry. It was observed that 6% of the oligosaccharides from the first glycosylation site of cardosin A are of the oligomannose type. Modified type glycans with proximal Fuc and without Xyl account for about 82%, 14% and 3% of the total oligosaccharides from the first and the second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. Oligosaccharides with Xyl but without proximal Fuc were only detected in the latter proteinase (4%). Glycans with proximal Fuc and Xyl account for 6%, 86% and 92% of total oligosaccharides from the first and second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively.
Costa
J
J
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Ashford
D A
DA
Nimtz
M
M
Bento
I
I
Frazäo
C
C
Esteves
C L
CL
Faro
C J
CJ
Kervinen
J
J
Pires
E
E
Veríssimo
P
P
Wlodawer
A
A
Carrondo
M A
MA
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002240:Carbohydrate Sequence; D006031:Glycosylation; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*
D009844:Oligosaccharides; D010940:Plant Proteins; D016282:Aspartic Acid Endopeptidases