Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells

Abstract

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.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family

“…However, they do harbor significant differences, such as distinct trafficking pathways in the cell and difference in glycosylation pattern (i.e. ratio of high-mannose to complex glycans) at individual glycosylation sites [27][28][29]. Study and characterization of potential N-glycosylation sites in members of this family nonetheless, gives insight into conservation and divergence of glycosylation with respect to function.…”

“…There have been studies carried out on mouse tyrosinase and TRP-1 and human tyrosinase, where putative glycosylation sites in these proteins were removed sequentially and the ensuing effect on activity and/or fate of the protein was determined [28,30,31]. However, direct investigation of glycosylation sites of TRP-2 has not been performed.…”

The human rhabdomyosarcoma cell line TE671 – Towards an innovative production platform for glycosylated biopharmaceuticals

“…Even for proteins with structural similarities -as for a tyrosinase and a tyrosinase-related protein both expressed in B16 mouse myeloma cells [58] -differential N-glycosylation was already described in the literature. Since sialylation of recombinant proteins is known to crucially affect the serum halflife and consequently the pharmacologic product efficiency [7], sialylated glycans are usually requested for therapeutical applications.…”

Anti-Melanogenic Property of Geoditin A in Murine B16 Melanoma Cells

“…One major band for tyrosinase (80 kD) was revealed in untreated B16 cells while a second band for protein of smaller size (about 70 kD) increased in a dose-dependent manner in geoditin A-treated cells (Figure 6a). These bands represent different degrees of maturation of melanogenic proteins (tyrosinase and tyrosinase-related proteins) in the ER [27]. Nascent tyrosinase is glycosylated and properly folded in the ER and completely matured in the Golgi before it is secreted to melanosomes for melanogenesis.…”