Human LAMAN (lysosomal α-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 α-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 (Thr355→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+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).
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Research Article|
July 06 2004
Intracellular transport of human lysosomal α-mannosidase and α-mannosidosis-related mutants
Gaute HANSEN;
Gaute HANSEN
*Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway
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Thomas BERG;
Thomas BERG
*Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway
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Hilde M. F. RIISE STENSLAND;
Hilde M. F. RIISE STENSLAND
*Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway
†Department of Medical Genetics, University Hospital of Northern-Norway, N-9037 Tromsø, Norway
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Pirkko HEIKINHEIMO;
Pirkko HEIKINHEIMO
‡Department of Physical Chemistry, University of Tromsø, N-9037 Tromsø, Norway
§Department of Biochemistry and Food Chemistry, Vatselankatu 2, FIN-20014, University of Turku, Finland
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Helle KLENOW;
Helle KLENOW
†Department of Medical Genetics, University Hospital of Northern-Norway, N-9037 Tromsø, Norway
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Gry EVJEN;
Gry EVJEN
*Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway
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Øivind NILSSEN;
Øivind NILSSEN
†Department of Medical Genetics, University Hospital of Northern-Norway, N-9037 Tromsø, Norway
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Ole K. TOLLERSRUD
Ole K. TOLLERSRUD
1
*Department of Medical Biochemistry, University of Tromsø, N-9037 Tromsø, Norway
1To whom correspondence should be addressed (e-mail olekt@fagmed.uit.no).
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Publisher: Portland Press Ltd
Received:
October 01 2003
Revision Received:
March 22 2004
Accepted:
March 23 2004
Accepted Manuscript online:
March 23 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 381 (2): 537–546.
Article history
Received:
October 01 2003
Revision Received:
March 22 2004
Accepted:
March 23 2004
Accepted Manuscript online:
March 23 2004
Citation
Gaute HANSEN, Thomas BERG, Hilde M. F. RIISE STENSLAND, Pirkko HEIKINHEIMO, Helle KLENOW, Gry EVJEN, Øivind NILSSEN, Ole K. TOLLERSRUD; Intracellular transport of human lysosomal α-mannosidase and α-mannosidosis-related mutants. Biochem J 15 July 2004; 381 (2): 537–546. doi: https://doi.org/10.1042/BJ20031499
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