Molecular Cloning and Expression of a Third Type of Rabbit GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase

Abstract

Recent molecular investigation revealed that two closely related structural genes encode distinct GDP-Lfucose:␤-D-galactoside 2-␣-L-fucosyltransferases (␣1,2-fucosyltransferases). Some human cancer cells or tissues may express an aberrant ␣1,2-fucosyltransferase other than H-and Secretor-type ␣1,2-fucosyltransferase. However, definite evidence of the existence of a third type of ␣1,2-fucosyltransferase has not been demonstrated. Here we report the molecular cloning of a third type of rabbit ␣1,2-fucosyltransferase (RFT-III) from a rabbit genomic DNA library. The DNA sequence included an open reading frame coding for 347 amino acids, and the deduced amino acid sequence of RFT-III showed 59 and 80% identity with those of the previously reported two types of rabbit ␣1,2-fucosyltransferase, RFT-I and RFT-II, respectively. COS-7 cells transfected with the RFT-III gene exhibited ␣1,2-fucosyltransferase activity toward phenyl-␤-Gal as a substrate. Neuro2a (a murine neuroblastoma cell line) cells transfected with the RFT-III gene expressed fucosyl G M1 (type 3 H) but not Ulex europaeus agglutinin-1 lectin reactive antigens (type 2 H). Kinetic studies revealed that RFT-III exhibits higher affinity to types 1 (Gal␤1, 3GlcNAc) and 3 (Gal␤1, 3Gal-NAc) than to type 2 (Gal␤1, 4GlcNAc) oligosaccharides, which suggests that RFT-III as well as RFT-II is a Secretor-type ␣1,2-fucosyltransferase. RFT-III was expressed in the adult gastrointestinal tract. The RFT-I, -II, and -III genes were assigned within 90 kilobases on pulsed field gel electrophoresis analysis. These results constitute direct evidence that, at least in one mammalian species, three active ␣1,2-fucosyltransferases exist.1 catalyzes the fucosylation of terminal ␤-DGal residues and synthesizes H antigens. The activity of ␣1,2-fucosyltransferase was detected in various tissues and body fluids of mammals that had several different kinetic characteristics (1-5). In humans, genetic and biochemical studies have indicated that two distinct but closely linked structural genes (H and Se) code ␣1,2-fucosyltransferases with tissue-specific patterns (1, 6). The human H gene controls the expression of H (Fuc␣1,2Gal␤) antigens (along with A or B antigens or both) on erythrocytes, whereas the Se gene determines the soluble A, B, and H antigens in secretory glands, and Lewis b blood group antigens on red cells (for review, see Ref. 7). Homozygosity for null alleles for the H and Se genes yields the rare Bombay blood type and non-Secretor phenotype, respectively. Recent molecular cloning of the H and Se genes provided the molecular basis for the Bombay and para-Bombay blood types and the nonSecretor phenotype, respectively, revealing point mutations within the coding regions that abolish the ␣1,2-fucosyltransferase activity (8 -10). On the other hand, the Lewis phenotyping of erythrocytes and secretory glands revealed the Le(aϩbϩ) and partial Secretor phenotypes in selected Polynesian and Asian individuals (11, 12). These phenotypes, which are virtually absent in Caucasians, are tho...

Structure and Expression of H-type GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase Gene (FUT1)

“…The expression of these antigens in the digestive mucosa has been observed from amphibians to higher mammals, while only human and some higher anthropoid primates but not old world monkeys express these antigens on erythrocyte membranes. Recently, rabbit homologues of human FUT1, FUT2, and a pseudogene of FUT2 (Sec1) have been isolated (38,39). In addition, we have had indications that the old-world African green monkey had three functional ␣(1,2)fucosyltransferase genes that were homologous to human FUT1, FUT2 and Sec1.…”

Novel Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens

“…1a) are synthesized through sequential action of the α1,2-and α1,3\4-FucTs through H determinants (Avent, 1997 ;Watkins, 1995). However, unusual α1,2-FucT activity that synthesizes Le b from Le a or Le Y from Le X has been found in some human cancer cells or tissues (Blaszczyk-Thurin et al, 1988 ;Yazawa et al, 1993), and recently such an unusual (the third type) α1,2-FucT was also found in the normal cells of rabbit (Hitoshi et al, 1996).…”

Molecular Cloning, Genomic Mapping, and Expression of TwoSecretor Blood Group α(1,2)Fucosyltransferase Genes Differentially Regulated in Mouse Uterine Epithelium and Gastrointestinal Tract

“…In rabbits, three ␣(1,2)fucosyltransferases with sequence similarity to the human loci each demonstrate enzymatic activity in expression constructs, including rabbit SEC1 (45,46). Although the chromosomal locations of the rabbit ␣(1,2)fucosyltransferase loci have not been mapped, all three ␣(1,2)fuco- syltransferase loci are contained on a single 90-kb genomic segment in rabbits (46).…”

“…In rabbits, three ␣(1,2)fucosyltransferases with sequence similarity to the human loci each demonstrate enzymatic activity in expression constructs, including rabbit SEC1 (45,46). Although the chromosomal locations of the rabbit ␣(1,2)fucosyltransferase loci have not been mapped, all three ␣(1,2)fuco- syltransferase loci are contained on a single 90-kb genomic segment in rabbits (46). In this study, the three mouse ␣(1,2)fucosyltransferase loci are conserved with similar spacing and order between humans and mice (FUT1, FUT2, SEC1), consistent with gene duplication of an ancestral ␣(1,2)fucosyltransferase gene into FUT1 and FUT2, followed by a second duplication of FUT2 producing SEC1 (47).…”