A1 Refereed original research article in a scientific journal
Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants
Authors: Hansen G, Berg T, Stensland HMFR, Heikinheimo P, Klenow H, Evjen G, Nilssen O, Tollersrud OK
Publisher: PORTLAND PRESS
Publication year: 2004
Journal:: Biochemical Journal
Journal name in source: BIOCHEMICAL JOURNAL
Journal acronym: BIOCHEM J
Volume: 381
First page : 537
Last page: 546
Number of pages: 10
ISSN: 0264-6021
DOI: https://doi.org/doi:10.1042/BJ20031499
Abstract
Human LAMAN (lysosomal alpha-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 alpha-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 (Thr(355) --> Pro), 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+ (His(72) and Asp(196)), stabilizing the active-site nucleophile (Arg(220)) or positioning the active-site residue Asp(319) (Phe(320)).
Human LAMAN (lysosomal alpha-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 alpha-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 (Thr(355) --> Pro), 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+ (His(72) and Asp(196)), stabilizing the active-site nucleophile (Arg(220)) or positioning the active-site residue Asp(319) (Phe(320)).
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