title: Mutation of the glycosylated asparagine residue 286 in human CLN2 protein results in loss of enzymatic activity.
abstract: 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.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Hospital Hamburg Eppendorf, Germany.
authors.forename: Kostas
authors.initials: K
authors.lastname: Tsiakas
authors.forename: Robert
authors.initials: R
authors.lastname: Steinfeld
authors.forename: Stephan
authors.initials: S
authors.lastname: Storch
authors.forename: Junji
authors.initials: J
authors.lastname: Ezaki
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Lukacs
authors.forename: Eiki
authors.initials: E
authors.lastname: Kominami
authors.forename: Alfried
authors.initials: A
authors.lastname: Kohlschütter
authors.forename: Kurt
authors.initials: K
authors.lastname: Ullrich
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
mesh_terms: D000626:Aminopeptidases; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009472:Neuronal Ceroid-Lipofuscinoses / Q000201:enzymology / Q000235:genetics; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D057057:Serine Proteases; D013329:Structure-Activity Relationship; D000091346:Tripeptidyl-Peptidase 1
chemical_list: D000091346:Tripeptidyl-Peptidase 1; D001216:Asparagine; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human