Document Detail

Possible interference between tissue-non-specific alkaline phosphatase with an Arg54-->Cys substitution and acounterpart with an Asp277-->Ala substitution found in a compound heterozygote associated with severe hypophosphatasia.
MedLine Citation:
PMID:  10839996     Owner:  NLM     Status:  MEDLINE    
Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.
M Fukushi-Irié; M Ito; Y Amaya; N Amizuka; H Ozawa; S Omura; Y Ikehara; K Oda
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  348 Pt 3     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  2000 Jun 
Date Detail:
Created Date:  2000-08-03     Completed Date:  2000-08-03     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  633-42     Citation Subset:  IM    
Department of Oral Biochemistry, Faculty of Dentistry, Niigata University, Gakkocho-dori, Niigata 951-8514, Japan.
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MeSH Terms
Alanine / chemistry
Alkaline Phosphatase / chemistry,  genetics,  metabolism*
Amino Acid Substitution*
Arginine / chemistry
Aspartic Acid / chemistry
COS Cells
Cysteine / chemistry
DNA, Complementary
Hypophosphatasia / enzymology*,  genetics
Mutation, Missense
Reg. No./Substance:
0/DNA, Complementary; 52-90-4/Cysteine; 56-41-7/Alanine; 56-84-8/Aspartic Acid; 74-79-3/Arginine; EC Phosphatase

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