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

Abstract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal ␣(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three ␣(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1 demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an ␣(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2 mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2 expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fuc␣132Gal␤ epitopes proposed to mediate blastocyst adhesion.Implantation competence of uterine epithelium is hormonedependent (1) and is accompanied by morphological and biochemical changes in the luminal surface of this epithelium (2, 3). Although a variety of molecules have been implicated in the series of adhesive events leading to implantation, including fibronectin and laminin and their integrin counter-receptors (4 -11), cell surface heparin sulfate proteoglycans (12-16), and mucins (17), there is also compelling experimental evidence that specific cell surface glycoconjugates mediate the initial adhesive process (18 -22). Specifically, the terminal ␣(1,2)fucosylated oligosaccharide epitope known as the H type 1 moiety undergoes differential expression in the endometrium and early embryo, exhibits regulation during the mouse estrus cycle, and is present at a time appropriate for implantation (23)(24)(25). The epitope is also present on uterine epithelium of ovariectomized mice only after estrogen injection at a time correlated with endometrial receptivity (24). In addition, exogenous H type 1 oligosaccharide and monoclonal antibodies toward this epitope inhibit blastocyst adhesion in vitro, whereas isomeric oligosaccharides and isotype-controlled antibodies do not inhibit this attachment (25). Furthermore, a potential counter-receptor on hatched mouse blastocysts has been demonstrated by specific binding of fluorescently labeled H type 1 oligosaccharide (26,27). This binding was specific to the apical surface of mural trophectoderm where the initial adhesion to uterine epithelium is seen in the mouse. The glycosidic linkages of the H type 1 epitope, like other oligosacchari...

Deficiency of Reproductive Tract α(1,2)Fucosylated Glycans and Normal Fertility in Mice with Targeted Deletions of the FUT1 or FUT2 α(1,2)Fucosyltransferase Locus

“…Southern blot probes derived from positions 5Ј and 3Ј to the borders of the targeting constructs were used in primary and secondary screening of ES colonies. For FUT2, a 5Ј 400-bp BamHI/ KpnI probe and 3Ј 390-bp KpnI/SacI probe were derived from genomic clones previously mapped 5Ј and 3Ј to FUT2 (9). For FUT1, PCR was used to generate a 3Ј 355-bp probe (25-mer forward primer, 5Ј-CGC TTG TCT ATT TAG GAC AGG AAC C-3Ј; 25-mer reverse primer, 5Ј-GAT TCA GGA GCC AGA ACT GAT TCT C-3Ј) and a 5Ј 238-bp probe (22-mer forward primer, 5Ј-GTG GAA ACC CAA AAA GAA ACA G-3Ј; 20-mer reverse primer, 5Ј-CGT CTG CCT CAG AAG GAC TC-3).…”

“…FUT1 and FUT2 genomic DNA clones were isolated and mapped from a commercially available P1 genomic library (strain 129P2/ OlaHsd; Incyte Genomics, St. Louis, Mo.) as previously described (9). Targeting vectors were derived from the ␤-galactoside-containing nucleus-localizing plasmid pnlacF (49).…”

“…Dynamic control of fucosyltransferase activity is in turn apparently controlled by the dynamic modulation of the level of transcripts derived from one or more ␣(1,2)fucosyltransferase genes (9,40). In the uterine epithelium, control of ␣(1,2)fucosylated glycan expression largely correlates with cycle-dependent modulation of expression of the FUT2 ␣(1,2)fucosyltransferase locus (9).…”

“…Dynamic control of fucosyltransferase activity is in turn apparently controlled by the dynamic modulation of the level of transcripts derived from one or more ␣(1,2)fucosyltransferase genes (9,40). In the uterine epithelium, control of ␣(1,2)fucosylated glycan expression largely correlates with cycle-dependent modulation of expression of the FUT2 ␣(1,2)fucosyltransferase locus (9). In oophorectomized mice, estrogen activates the accumulation of uterine epithelial cell ␣(1,2)fucosyltransferase activity and its mRNA, whereas progesterone inhibits accumulation (19,40).…”

Molecular Cloning and Characterization of a Novel α1,2-Fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

“…The ileal epithelium of adult mice in normal growth cages is characterized by expression of glycoconjugates with the sequence Fuc␣1-2Gal-R; however, the intestines of germfree mice are deficient in such fucose-containing structures (35). Inoculation of the germ-free mice with Bacteroides thetaiotaomicron significantly stimulated expression of Fuc␣1-2Gal-R structures and transcripts encoding an ␣1,2FT (35), which could be derived from either SEC1 or FUT2 (36). It was recently shown in germ-free mice that administration of intestinal microbes stimulated expression of MFUT-II, the ortholog of human FUCT2 (Se) and rat FTB (37,38).…”