A Family of Human β4-Galactosyltransferases
CLONING AND EXPRESSION OF TWO NOVEL UDP-GALACTOSE:β-N-ACETYLGLUCOSAMINE β1,4-GALACTOSYLTRANSFERASES, β4Gal-T2 AND β4Gal-T3*
- Raquel Almeida‡§,
- Margarida Amado‡§,
- Leonor David§,
- Steven B. Levery¶,
- Eric H. Holmes‖,
- Gerard Merkx**,
- Ad Geurts van Kessel**,
- Eske Rygaard‡,
- Helle Hassan‡,
- Eric Bennett‡ and
- Henrik Clausen‡‡
- From the ‡School of Dentistry, University of Copenhagen, Nørre Allé 20, DK-2200 Copenhagen N, Denmark, the§Institute of Molecular Pathology and Immunology of University of Porto, IPATIMUP, Rua Dr. R. Frias s/n, 4200 Porto, Portugal, the ¶Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, the ‖Department of Cell Surface Biochemistry, Northwest Hospital, Seattle, Washington 98125, and the **Department of Human Genetics, University Hospital, Nijmegen 6050 HB, The Netherlands
Abstract
BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:β-N-acetylglucosamine β1,4-galactosyltransferase, designated β4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of β4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with β4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of β4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:β-N-acetylglucosamine β1,4-galactosyltransferases, thus designated β4Gal-T2 and β4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with β4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple β4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to β4Gal-T1.
Footnotes
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↵* This work was supported by the Danish Cancer Society, the Ingeborg Roikjer Foundation, the Velux Foundation, the Danish Medical Research Council, the Lundbeck Foundation, Praxis XXI 2/2.1/BIA/276/94, and Grant 5 P41 RR05351 from the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y12509 and Y12510.
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↵‡ To whom the correspondence should be addressed. Tel.: 45-35326835; Fax: 45-35326505; E-mail:henrik.clausen{at}odont.ku.dk.
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↵1 The abbreviations used are: RT-PCR, reverse transcription-polymerase chain reaction; EST, expressed sequence tags; bp, base pair(s); Cer, ceramide.
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↵2 R. Almeida and H. Clausen, unpublished observation.
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↵3 M. Amado and H. Clausen, manuscript in preparation.
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- Received June 27, 1997.
- Revision received September 3, 1997.
