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A functional splice variant of the human Golgi CMP-sialic acid transporter

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Abstract

The human Golgi Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Lex antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

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Abbreviations

Ac4ManNAz:

N-azidoacetylmannosamine

C/shRNA:

Control/shRNA plasmid

CDG:

Congenital Disorder of Glycosylation

CHO:

Chinese hamster ovary cell line

CMP-Sia:

Cytidine-5′-monophospho-N-acetylneuraminic acid

ER:

Endoplasmic Reticulum

MO:

Morpholino

MO-Scrambled:

Control morpholino oligo

MO-Carboxy:

Carboxyfluorescein morpholino oligo

MO-CMPTR6:

Morpholino directed against exon 6

NST:

Nucleotide sugar transporter

PCR:

Polymerase chain reaction

PNA:

Peanut agglutinin

RCA-I:

Ricinus communis agglutinin I

SiaNAz:

N-azidoacetyl sialic acid

SLC35A1/shRNA:

SLC35A1/shRNA plasmid

WGA:

Wheat germ agglutinin

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Acknowledgments

We are grateful to Dr. Alexandra Vincent from Gene Tools for her help in designing the antisense oligos morpholinos. We also acknowledge the support of the synthesis unit of the Instituto de Biotecnología-Universidad Nacional Autónoma de México in the synthesis of antisense oligos for PCR.

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Authors and Affiliations

  1. Laboratorio de Glicobiología Humana y Diagnóstico Molecular, Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001,Col. Chamilpa, 62209 Cuernavaca, MOR, Cuernavaca, México

    Roberta Salinas-Marín & Iván Martínez-Duncker

  2. INSERM U1197, Paul Brousse Hospital, University of Paris Sud XI, 94800, Villejuif, France

    Rosella Mollicone

Authors
  1. Roberta Salinas-Marín
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  2. Rosella Mollicone
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  3. Iván Martínez-Duncker
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Correspondence to Iván Martínez-Duncker.

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The authors declare that they have no competing interests.

Funding

Work carried out in our laboratory was supported by CONACYT grant 57157. RSM was supported by CONACYT scholarship [217256]. IMD and RSM were supported by the Latin American Society for Glycobiology and the ECOS-CONACYT-SEP-ANUIES grant M09-S03. Red Temática Glicociencia en Salud- CONACYT grant 253596.

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Salinas-Marín, R., Mollicone, R. & Martínez-Duncker, I. A functional splice variant of the human Golgi CMP-sialic acid transporter. Glycoconj J 33, 897–906 (2016). https://doi.org/10.1007/s10719-016-9697-8

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  • DOI: https://doi.org/10.1007/s10719-016-9697-8

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