title: C-terminus glycans with critical functional role in the maturation of secretory glycoproteins.
abstract: The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs--one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.
authors.affiliation: Department Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Bucharest, Romania.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cioaca
authors.forename: Simona
authors.initials: S
authors.lastname: Ghenea
authors.forename: Laurentiu N
authors.initials: LN
authors.lastname: Spiridon
authors.forename: Marioara
authors.initials: M
authors.lastname: Marin
authors.forename: Andrei-Jose
authors.initials: AJ
authors.lastname: Petrescu
authors.forename: Stefana M
authors.initials: SM
authors.lastname: Petrescu
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D014442:Monophenol Monooxygenase