Senescence marker protein 30 functions as gluconolactonase in l-ascorbic acid biosynthesis, and its knockout mice are prone to scurvy

doi:10.1073/pnas.0511225103

Senescence marker protein 30 functions as gluconolactonase in l-ascorbic acid biosynthesis, and its knockout mice are prone to scurvy

*Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan;
^†Vascular Medicine and Geriatrics, Tokyo Medical and Dental University, Tokyo 113-8510, Japan;
^§Department of Biochemistry, Wakayama Medical University, Wakayama 641-0012, Japan; and
^¶Department of Biochemistry, Toho University, Chiba 274-8510, Japan

Edited by John E. Halver, University of Washington, Seattle, WA, and approved February 27, 2006
↵ ^‡Y.K., Y.I., and Y.S. contributed equally to this work. (received for review December 30, 2005)

Abstract

We originally identified senescence marker protein 30 (SMP30) as a distinctive protein whose expression decreases in an androgen-independent manner with aging. Here, we report its sequence homology found in two kinds of bacterial gluconolactonases (GNLs) by using the blast search. Then, through a biochemical study, we identify SMP30 as the lactone-hydrolyzing enzyme GNL of animal species. SMP30 purified from the rat liver had lactonase activity toward various aldonolactones, such as d- and l-glucono-δ-lactone, d- and l-gulono-γ-lactone, and d- and l-galactono-γ-lactone, with a requirement for Zn²⁺ or Mn²⁺ as a cofactor. Furthermore, in SMP30 knockout mice, no GNL activity was detectable in the liver. Thus, we conclude that SMP30 is a unique GNL in the liver. The lactonase reaction with l-gulono-γ-lactone is the penultimate step in l-ascorbic acid (AA) biosynthesis, and the essential role of SMP30 in this synthetic process was verified here by a nutritional study using SMP30 knockout mice. These knockout mice (n = 6), fed a vitamin C-deficient diet, did not thrive; i.e., they displayed symptoms of scurvy such as bone fracture and rachitic rosary and then died by 135 days after the start of receiving the deficient diet. The AA levels in their livers and kidneys at the time of death were <1.6% of those in WT control mice. In addition, by using the SMP30 knockout mouse, we demonstrate that the alternative pathway of AA synthesis involving d-glucurono-γ-lactone operates in vivo, although its flux is fairly small.

Footnotes

^‖To whom correspondence may be addressed. E-mail: nishikim{at}wakayama-med.ac.jp
**To whom correspondence may be addressed at:
Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
E-mail: ishigami{at}tmig.or.jp
Author contributions: Y.K., Y.I., Y.S., K.S., S.G., M.N., N.M., and A.I. designed research; Y.K., Y.I., Y.S., S.H., S.K., M.N., and A.I. performed research; Y.K., Y.I., Y.S., S.H., S.K., K.S., S.G., M.N., N.M., and A.I. contributed new reagents/analytic tools; Y.K., Y.I., Y.S., S.H., S.K., K.S., S.G., M.N., N.M., and A.I. analyzed data; and Y.K., Y.I., Y.S., K.S., M.N., N.M., and A.I. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

AA,

l-ascorbic acid;

BMD,

bone mineral density;

GNL,

gluconolactonase;

MBP,

maltose-binding protein;

SMP30,

senescence marker protein 30.