Glycobiology Advance Access originally published online on January 21, 2004
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Glycobiology vol 14 no 4 pp. 1C-5C, 2004
Glycobiology vol. 14 no. 4 © Oxford University Press 2004; all rights reserved.
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Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity
2 Department of Biochemistry, Children's Hospital, University Hospital Hamburg Eppendorf, Martinistraße 52, Bldg W23, D20246 Hamburg, Germany; and 3 Department of Biochemistry, Juntendo University School of Medicine; Tokyo 113, Japan
Received on July 25, 2003; revised on December 11, 2003; accepted on January 2, 2004
Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by the deficiency of the lysosomal tripeptidyl peptidase-I encoded by CLN2. We previously detected in two LINCL patients a homozygous missense mutation, p.Asn286Ser, that affects a potential N-glycosylation site. We introduced the p.Asn286Ser mutation into the wild-type CLN2 cDNA and performed transient expression analysis to determine the effect on the catalytic activity, intracellular targeting, and glycosylation of the CLN2 protein. Expression of mutant p.Asn286Ser CLN2 in HEK293 cells revealed that the mutant was enzymatically inactive. Western blot analysis demonstrated that at steady state the amounts of expressed p.Asn286Ser CLN2 were reduced compared with wild-type expressing cells. The rate of synthesis and the sorting of the newly synthesized p.Asn286Ser CLN2 in the Golgi was not affected compared with wild-type CLN2 protein. The electrophoretic mobility of the immunoprecipitated mutant p.Asn286Ser CLN2 was increased by approximately 2 kDa compared with the wild-type CLN2 protein, whereas deglycosylation led to the generation of polypeptides of the same apparent size. The data suggest that mutant p.Asn286Ser CLN2 lacks one oligosaccharide chain resulting in enzymatic inactivation.
1 To whom correspondence should be addressed; e-mail: braulke{at}uke.uni-hamburg.de
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