J. Biochem, 2003, Vol. 134, No. 1 63-70
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Tissue-Nonspecific Alkaline Phosphatase with an Asp289
Val Mutation Fails to Reach the Cell Surface and Undergoes Proteasome-Mediated Degradation
1 Division of Biochemistry and 2 Removable Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514; 3 Kitasato Junior College of Health and Hygienic Sciences, 500 Kurotsuchi Shinden, Yamatomachi, Minamiuonuma-gun, Niigata 929-7241; and 4 Center for Transdisciplinary Research, Niigata University
A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171–172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-ß-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu218
Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca2+ coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.
+ To whom correspondence should be addressed. E-mail: oda{at}dent.niigata-u.ac.jp
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