N-glycosylation of carnosinase influences protein secretion and enzyme activity: implications for hyperglycemia

Diabetes. 2010 Aug;59(8):1984-90. doi: 10.2337/db09-0868. Epub 2010 May 11.

Abstract

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.

MeSH terms

  • Adult
  • Age of Onset
  • Aged
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Diabetes Mellitus / enzymology
  • Diabetes Mellitus / epidemiology
  • Diabetes Mellitus / genetics
  • Diabetic Nephropathies / enzymology
  • Diabetic Nephropathies / genetics*
  • Dipeptidases / drug effects
  • Dipeptidases / genetics*
  • Dipeptidases / metabolism*
  • Gene Expression Regulation, Enzymologic
  • Genetic Predisposition to Disease*
  • Genotype
  • Glycosylation
  • Hexosamines / metabolism
  • Humans
  • Hyperglycemia / enzymology
  • Hyperglycemia / metabolism*
  • Middle Aged
  • Mutagenesis, Site-Directed
  • Polymorphism, Genetic*
  • Reference Values
  • Transfection
  • Tunicamycin / pharmacology

Substances

  • Hexosamines
  • Tunicamycin
  • Dipeptidases
  • aminoacyl-histidine dipeptidase