Translation Rate of Human Tyrosinase Determines ItsN-Linked Glycosylation Level

Abstract

Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-GlyThr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.

Tyrosinase Maturation and Oligomerization in the Endoplasmic Reticulum Require a Melanocyte-specific Factor

“…1A, bottom panel). The doublet corresponds to TYR possessing all seven (TYR 7 ) or six (TYR 6 ) N-linked glycans (12). As reported previously, the fraction of TYR 6 was increased in the faster-translating RRL system compared with the slower WG translation, where each form was present in equal amounts.…”

“…We have shown previously that ER translocation and co-translational glycosylation of TYR can be monitored in a cell-free system composed of an in vitro translation system coupled with canine pancreas ER-derived microsomes (12). In these studies, replacing the native signal sequence of TYR with that of the murine major histocompatibility complex class I molecule K b (K b SS) increased the efficiency of translation and translocation by severalfold.…”

“…The construction of the plasmid encoding WT TYR, termed pSP72-K b SS/TYR, has been described previously (12). To generate the mutant TYR forms C89R and C500S, the corresponding Cys in pSP72-K b SS/ TYR were changed to Arg and Ser, respectively, using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

“…Radioactive 35 Slabeled TYR was translated for 1 h at 27°C with RRL or WG in the presence of canine pancreas microsomes or semipermeabilized cells. Translation reactions were carried out as described previously (12). In some experiments, the samples were alkylated either with N-ethylmaleimide (20 mM) (23) or with 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid (AMS; 14 mM) in 80 mM Tris, pH 6.8, 1% SDS to block free sulfhydryls.…”

“…Although all seven sites are utilized, one site (Asn 290 ) is inefficiently processed due to an adjacent Pro residue (AsnGly-Thr-Pro) (12). The presence of this inefficient glycosylation site, which is absent in mouse tyrosinase (Tyr), creates a heterogeneous population of TYR possessing six or seven glycans.…”