Elsevier

Molecular Genetics and Metabolism

Volume 77, Issues 1–2, September–October 2002, Pages 99-107
Molecular Genetics and Metabolism

Unraveling the molecular pathogenesis of free sialic acid storage disorders: altered targeting of mutant sialin

https://doi.org/10.1016/S1096-7192(02)00124-5Get rights and content

Abstract

Salla disease (SD) and infantile sialic acid storage disease (ISSD) are recessively inherited, neuro-degenerative disorders caused by mutations in the SLC17A5 gene. The gene product, sialin, is a lysosomal membrane protein which transports free sialic acid across the membrane. Although the function of sialin is basically known, the details of biosynthesis and intracellular trafficking as well as functional consequences of disease mutations in the SLC17A5 gene are not characterized. Here we studied for the first time the expression, localization, and targeting of the wild-type sialin as well as two mutant polypeptides; one mimicking the Finnish founder mutation, R39C (SallaFIN), and the other a deletion (del268–272) found in ISSD patients using in vitro expression of the corresponding cDNA constructs. The wild-type sialin was targeted to lysosomes whereas a significant fraction of the SallaFIN polypeptides and the majority of the ISSD polypeptides remained in the Golgi compartment. Further, using a temperature block of intracellular transport, we observed that the rate of the trafficking of the mutant polypeptides to lysosomes is significantly slower than that of their wild-type counterpart. These findings are in line with the phenotypic differences between SD and ISSD, the former presenting mental retardation with long life span in contrast to the latter being an early fatal disorder.

Introduction

Lysosomal free sialic acid storage diseases (OMIM 269920), Salla disease (SD) and infantile sialic acid storage disease (ISSD), are recessively inherited allelic neuro-degenerative disorders [1]. The defective gene, SLC17A5, coding for the putative lysosomal sialic acid transporter was positionally cloned and so far altogether 20 different disease causing mutations have been identified [2], [3]. SD is enriched in the isolated Finnish population and a founder mutation, SallaFIN, changing a conserved arginine into a cysteine (R39C) is found in all Finnish patients. The carrier frequency varies from 1:50 to 1:200 being highest in the northeastern region of Finland. ISSD patients carry a wide spectrum of mutations with no known ethnic clustering. In addition, there are rare compound heterozygotes who present an intermediate phenotype with SallaFIN in one allele and another mutation in the other allele. Some genotype–phenotype correlation exists since the phenotype associated with the homozygote R39C mutation is milder than the disease outcome in patients with other mutations [3].

The SLC17A5 gene mutated in both Salla disease and ISSD codes for a 495 amino acid protein designated sialin. Sialin is an integral membrane protein predicted to have 12 transmembrane (TM) domains with both amino- and carboxyterminal ends on the cytosolic side of the membrane [2]. Although the function of the protein is known, it is unclear how mutations in the sialin gene exert their effect at the cellular level. To explore the intracellular localization and trafficking of the wild-type and mutant sialin we expressed the wild-type sialin cDNA and two mutant constructs in HeLa, BHK, and COS-1 cells. The mutant cDNAs were constructed to mimic the SallaFIN mutation and a five-amino acid deletion (del SSRLN) found in an ISSD patient. We observed that the wild-type sialin was transported to the lysosomal membrane in 1 h whereas the trafficking of both mutants from the ER-Golgi intermediate compartment (ERGIC) to lysosomes was severely hampered.

Section snippets

Construction of expression plasmids and site directed mutagenesis

The coding region of the SLC17A5 cDNA (GenBank Accession No. XM_004396) was PCR-amplified from reverse transcribed human lymphoblastoid total RNA (3-primer: 5-GCGATGGAATTCTCAGTGTCTGTGTCCATGGTG-3 and 5-primer: CATCGCGAATTCGGCGTCATGAGGTCTCCGGT-3). The PCR-product was cleaved with the restriction enzyme EcoRI (New England Biolabs, Beverly, MA) and ligated into a pSvPoly expression vector [4] cleaved with the same enzyme. The insert was sequenced using the ABI Prism Big Dye Terminator Cycle

The apparent molecular weight of in vitro translated sialin is smaller than the calculated mass and the protein expressed in cells

The positions of the SallaFIN mutation and the ISSD deletion are shown in the schematic presentation of sialin polypeptide in Fig. 1. The SallaFIN mutation (R39C) precedes the first transmembrane domain and the five amino acid ISSD deletion (del268–272) resides in the third cytosolic loop of the predicted topology model of sialin. The amino acids (251–265) corresponding to the synthetic peptide used for antibody production are located in the same loop (Fig. 1). Neither of the studied mutations

Discussion

Sialin is a transmembrane protein which functions as a transporter of free sialic acid across the lysosomal membrane [2]. This transporter also recognizes other acidic sugars (e.g., glucuronic and iduronic acid) and some aliphatic monocarboxylated anions like l-lactate [10], [11]. Free sialic acid is produced by lysosomal sialidase induced degradation of glycoproteins and glycolipids in lysosomes. The defective function of sialin leads to accumulation of free sialic acid in lysosomes and

Acknowledgements

We thank Paula Hakala for her excellent technical assistance. This study was supported financially by the Academy of Finland and the Pediatric Research Foundation (Ulla Hjelt Fund).

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