title: Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.
abstract: Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).
authors.affiliation: Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway.
authors.forename: Gaute
authors.initials: G
authors.lastname: Hansen
authors.forename: Thomas
authors.initials: T
authors.lastname: Berg
authors.forename: Hilde M F
authors.initials: HM
authors.lastname: Riise Stensland
authors.forename: Pirkko
authors.initials: P
authors.lastname: Heikinheimo
authors.forename: Helle
authors.initials: H
authors.lastname: Klenow
authors.forename: Gry
authors.initials: G
authors.lastname: Evjen
authors.forename: Øivind
authors.initials: Ø
authors.lastname: Nilssen
authors.forename: Ole K
authors.initials: OK
authors.lastname: Tollersrud
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000378:metabolism; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005838:Genotype; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010641:Phenotype; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000379:methods; D043323:alpha-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008363:alpha-Mannosidosis / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D008361:Mannosidases; D043323:alpha-Mannosidase