N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase.
Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.
Wang
Yunling
Y
Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, S-141 86 Huddinge, Sweden.
Norgård
Maria
M
Andersson
Göran
G
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D005269:Femur / Q000201:enzymology*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D018411:Spodoptera / Q000201:enzymology*; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C091137:purple acid phosphatase; D000135:Acid Phosphatase