Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells.
Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.
Negroiu
G
G
Institute of Biochemistry, Romanian Academy, Splaiul Independentei 296, 77700 Bucharest 17, Romania.
Branza-Nichita
N
N
Petrescu
A J
AJ
Dwek
R A
RA
Petrescu
S M
SM
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D009844:Oligosaccharides / Q000032:analysis; D010088:Oxidoreductases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry; D011506:Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011506:Proteins; D017485:1-Deoxynojirimycin; C059896:miglustat; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase