Tài liệu Báo cáo khoa học: Novel aggregate formation of a frame-shift mutant protein of

Novel aggregate formation of a frame-shift mutant protein

of tissue-nonspecific alkaline phosphatase is ascribed

to three cysteine residues in the C-terminal extension

Retarded secretion and proteasomal degradation

Keiichi Komaru1,2, Yoko Ishida1

, Yoshihiro Amaya1

, Masae Goseki-Sone3

, Hideo Orimo4

and Kimimitsu Oda1,5

1 Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, Gakkocho-dori, Niigata, Japan

2 Kitasato Junior College of Health and Hygienic Sciences, Yamatomachi, Minami-Uonuma-shi, Niigata, Japan

3 Department of Food and Nutrition, Japan Women’s University, Mejirodai, Bunkyo-ku, Tokyo, Japan

4 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan

5 Center for Transdisciplinary Research, Niigata University, Japan

Keywords

aggregation; alkaline phosphatase;

degradation; hypophosphatasia; proteasome;

ubiquitin

Correspondence

K. Oda, Division of Biochemistry, Course for

Oral Life Science, Niigata University,

Graduate School of Medical and Dental

Sciences, 2–5274, Gakkocho-dori, Niigata,

951–8514, Japan

Fax: +81 25 227 2831

Tel: +81 25 227 2827

E-mail: [email protected]

(Received 21 December 2004, revised 30

January 2005, accepted 3 February 2005)

doi:10.1111/j.1742-4658.2005.04597.x

In the majority of hypophosphatasia patients, reductions in the serum lev￾els of alkaline phosphatase activity are caused by various missense muta￾tions in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A

unique frame-shift mutation due to a deletion of T at cDNA number 1559

[TNSALP (1559delT)] has been reported only in Japanese patients with

high allele frequency. In this study, we examined the molecular phenotype

of TNSALP (1559delT) using in vitro translation ⁄translocation system and

COS-1 cells transiently expressing this mutant protein. We showed that the

mutant protein not only has a larger molecular size than the wild type

enzyme by
12 kDa, reflecting an 80 amino acid-long extension at its

C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In

support of this, alkaline phosphatase activity of the cells expressing TNS￾ALP (1559delT) was localized at the juxtanucleus position, but not on the

cell surface. However, only a limited amount of the newly synthesized pro￾tein was released into the medium and the rest was polyubiquitinated,

followed by degradation in the proteasome. SDS ⁄ PAGE and analysis by

sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms

a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggre￾gate form of TNSALP (1559delT) exhibited a significant enzyme activity.

When all three cysteines at positions of 506, 521 and 577 of TNS￾ALP (1559delT) were replaced with serines, the aggregation disappeared

and instead this modified mutant protein formed a noncovalently associ￾ated dimer, strongly indicating that these cysteine residues in the C-ter￾minal region are solely responsible for aggregate formation by cross-linking

the catalytically active dimers. Thus, complete absence of TNSALP on cell

surfaces provides a plausible explanation for a severe lethal phenotype of a

homozygote hypophosphatasia patient carrying TNSALP (1559delT).

Abbreviations

Bz-Asn-Gly-Thr-NH2, benzoyl-asparagine-glycine-threonine-amide; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum;

ECL, enhanced chemiluminescence; GPI, glycosylphosphatidylinositol; LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal); LLM, N-acetyl-L￾leucinyl-L-leucinyl-L-methional); MG-132, benzyloxycarbonyl-L-leucinyl-L-leucinyl-L-leucinal; PI-PLC, phosphatidylinositol-specific phospholipase

C; PNGase F, peptide:N-glycosidase F; MEM, minimum essential medium; TNSALP, tissue-nonspecific alkaline phosphatase; sTNSALP,

soluble form of TNSALP.

1704 FEBS Journal 272 (2005) 1704–1717 ª 2005 FEBS