N-glycosylation of carnosinase influences protein secretion and enzyme activity: implications for hyperglycemia.
OBJECTIVE
The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion. Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1 secretion is changed under hyperglycemic conditions.


RESULTS
N-glycosylation of CN-1 was either inhibited by tunicamycin in pCSII-CN-1-transfected Cos-7 cells or by stepwise deletion of its three putative N-glycosylation sites. CN-1 protein expression, N-glycosylation, and enzyme activity were assessed in cell extracts and supernatants. The influence of hyperglycemia on CN-1 enzyme activity in human serum was tested in homozygous (CTG)(5) diabetic patients and healthy control subjects. Tunicamycin completely inhibited CN-1 secretion. Deletion of all N-glycosylation sites was required to reduce CN-1 secretion efficiency. Enzyme activity was already diminished when two sites were deleted. In pCSII-CN-1-transfected Cos-7 cells cultured in medium containing 25 mmol/l d-glucose, the immature 61 kilodaltons (kDa) CN-1 immune reactive band was not detected. This was paralleled by an increased GlcNAc expression in cell lysates and CN-1 expression in the supernatants. Homozygous (CTG)(5) diabetic patients had significantly higher serum CN-1 activity compared with genotype-matched, healthy control subjects.


CONCLUSIONS
We conclude that apart from the (CTG)(n) polymorphism in the signal peptide of CN-1, N-glycosylation is essential for appropriate secretion and enzyme activity. Since hyperglycemia enhances CN-1 secretion and enzyme activity, our data suggest that poor blood glucose control in diabetic patients might result in an increased CN-1 secretion even in the presence of the (CTG)(5) allele.
Riedl
Eva
E
15th Medical Clinic, University Medical Centre Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
Koeppel
Hannes
H
Pfister
Frederick
F
Peters
Verena
V
Sauerhoefer
Sibylle
S
Sternik
Paula
P
Brinkkoetter
Paul
P
Zentgraf
Hanswalter
H
Navis
Gerjan
G
Henning
Robert H
RH
Van Den Born
Jacob
J
Bakker
Stephan J L
SJ
Janssen
Bart
B
van der Woude
Fokko J
FJ
Yard
Benito A
BA
D000328:Adult; D017668:Age of Onset; D000368:Aged; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003920:Diabetes Mellitus / Q000201:enzymology / Q000453:epidemiology / Q000235:genetics; D003928:Diabetic Nephropathies / Q000201:enzymology / Q000235:genetics*; D004150:Dipeptidases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006031:Glycosylation; D006595:Hexosamines / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000201:enzymology / Q000378:metabolism*; D008875:Middle Aged; D016297:Mutagenesis, Site-Directed; D011110:Polymorphism, Genetic; D012016:Reference Values; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D006595:Hexosamines; D014415:Tunicamycin; D004150:Dipeptidases; C019673:aminoacyl-histidine dipeptidase
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