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Functional characterization of wild-type and mutant human sialin.

ABSTRACT: The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.

SUBMITTER: Morin P

PROVIDER: S-EPMC533050 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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Functional characterization of wild-type and mutant human sialin.

Morin Pierre P Sagné Corinne C Gasnier Bruno B

The EMBO journal 20041028 23

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein ...[more]

PMID: 15510212

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Project description:Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients [Tuchman (1993) Hum. Mutat. 2, 174-178; Tuchman and Plante (1995) Hum. Mutat. 5, 293-295]. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) [Tuchman, Morizono, Reish, Yuan and Allewell (1995) J. Med. Genet. 32, 680-688], and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase [Villeret, Tricot, Stalon and Dideberg (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10762-10766], indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, Ndelta-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase [Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754-4761], suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 degrees C. Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.

Dataset Information

Functional characterization of wild-type and mutant human sialin.

Publications

Functional characterization of wild-type and mutant human sialin.

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