Oligosaccharides represent major components of animal cell surfaces and are believed to function in cellular interactions during development and differentiation(1, 2) , oncogenic transformation(3) , and inflammation(4) . Identification of specific oligosaccharide ligands for the selectin family of cell adhesion molecules directly links cell surface carbohydrates to cell-cell communication in the context of inflammatory response(5, 6, 7) . The proposed ligands for E-selectin and P-selectin are fucosylated oligosaccharides (for review, see (4) and (8) ), whose biosynthesis is catalyzed by -(1-3)-fucosyltransferases (1-3FTs). ()These enzymes are encoded by one or more distinct and tightly regulated 1-3FT genes.

Much of the interest in discovering functional roles for oligosaccharides during development is derived from studies documenting precise temporal-spatial expression patterns for some oligosaccharides during human and murine embryogenesis(9, 10, 11, 12) . The murine stage-specific embryonic antigen-1 (SSEA-1; (Gal1-4[Fuc1-3]GlcNAc; Lewis x)), for example, is expressed coincident with morula compaction at the 8-16 cell stage of the preimplantation mouse embryo(13, 14) . Since SSEA-1 structural analogs appear to inhibit compaction, it has been suggested that this antigen may participate in this process(1, 2) . While it has been proposed that the SSEA-1 determinant functions to promote homotypic adhesion(15) , neither the physiological relevance of this interaction during compaction nor the existence of other preimplantation-specific receptors for SSEA-1 have been demonstrated. Furthermore, it has not been possible to identify and directly demonstrate functional correlates for SSEA-1 expression patterns during early embryogenesis.

Virtually nothing is known about the molecular mechanisms that determine the tissue-specific and developmentally regulated expression patterns of the oligosaccharides implicated in morphogenic events during early murine embryogenesis. Expression of surface-localized SSEA-1 molecules may be regulated by differential expression of 1-3FT(s) required for their synthesis (16) and of sialyltransferases (17) and an -(1-3)-galactosyltransferase (18) that may mask SSEA-1 expression(9, 10, 18) . The relative contributions of these and other regulatory mechanisms, in the context of the developing embryo, remain undefined.

To begin to define, in detail, the enzymes and mechanisms that determine expression of Fuc1-3GlcNAc linkages in murine cell surface oligosaccharides, we have isolated and characterized a murine gene that corresponds to a human 1-3FT gene termed Fuc-TIV (Refs. 19 and 20; also known as ELFT for ELAM-1 ligand fucosyl transferase, (21) ). The mouse enzyme differs from human Fuc-TIV/ELFT in its relatively higher apparent affinity for -(2-3)-sialylated type acceptor substrates in vitro, and is able to efficiently synthesize an unusual tetrasaccharide (Gal1-3Gal1-4[Fuc1-3]GlcNAc) whose existence in periimplantation mouse tissues has been previously inferred(10) . Northern blot analyses confirm that transcripts corresponding to this gene accumulate in leukocytic cell lines and in leukocyte-rich tissues in the mouse. However, these analyses, and companion in situ hybridization studies, demonstrate that Fuc-TIV/ELFT transcripts are unexpectedly abundant in epithelial cells lining the gastrointestinal and reproductive tracts and suggest that this sequence, and the cognate Fuc1-3GlcNAc linkages whose expression it determines may have unexpected functions in these tissues.

EXPERIMENTAL PROCEDURES

Cell Culture

Antibodies

Murine Genomic Library Screening

Subcloning and DNA Sequence Analysis

Northern Blot Analysis

Figure 1: Nucleotide and deduced amino acid sequences of the murine 1-3FT gene and comparison to the human Fuc-TIV/ELFT gene and its cDNAs. The DNA sequence of the murine gene, and its predicted protein sequences, correspond to MFT-IV DNA, and MFT-IV AA, respectively. The DNA sequence of the human Fuc-TIV/ELFT gene, and predicted protein sequences, correspond to lines denoted by HFT-IV DNA, and HFT-IV AA, respectively. The A residues of the putative initiation codon of the murine 1-3FT gene and of the human ELFT/Fuc-TIV sequence are assigned as residue 1 of their nucleotide sequence. The methionine residues corresponding to these codons are assigned as residue 1 of the corresponding protein sequences. These positions are further denoted by an arrow pointing to these aligned initiator methionine codons (start of ELFT AA). The initiation codon for the ``long'' form of ELFT (21) is also indicated by arrows (start of ELFT AA; Initiator codon for ELFT-L protein). The initiator methionine for the long form of the murine polypeptide is also indicated by an arrow (start of ``long'' mouse polypeptide). This methionine codon is encompassed within the NcoI site (dottedunderlined) used to construct the vector pcDNAI-mFuc-TIV. Arrows also indicate the 5`-most residues found in the ELFT and ELFT-L cDNAs (21) (start of ELFT cDNA; start of ELFT-L cDNA). The sequence alignment was generated using the BESTFIT and GAP programs of the University of Wisconsin Genetics Computer Group(39) . The GAP program generates symbols between aligned amino acids, according to the evolutionary distance between them, as measured by Dayhoff (50) and normalized by Gribskov(51) . Amino acid sequence identities are assigned a score of 1.5, denoted by a verticalbar; related amino acid residues with scores from 0.5 to 1.4 are denoted by a two dots, less strongly related amino acid residues with scores between 0.1 to 0.4 are denoted by onedot; no symbol is placed between dissimilar amino acid pairs with scores less than 0.1. Gaps in the amino acid sequence alignment are denoted by a dash. Nucleotide sequence identity is indicated by verticallines between aligned corresponding residues. Gaps in the nucleotide sequence alignment are indicated by a dottedline. The predicted transmembrane domain of the murine enzyme (amino acids 53-74) is doubleunderlined. Consensus sites for asparagine-linked glycosylation are underlined.

Transfection and Expression of Murine Fuc-TIV Gene

Flow Cytometry Analysis

1-3FT Assays

Figure 3: pH- and Mn concentration-1-3FT activity profiles. A, pH-activity profiles. The enzymatic activity in COS-7 cells transfected with pcDNAI-mFuc-TIV or pcDNAI-hFuc-TIV (20) was assayed using 20 mMN-acetyllactosamine and 3 mM GDP-[C]fucose (see ``Experimental Procedures''). Reactions contained 50 mM sodium acetate, ; sodium phosphate, ; or Tris-HCl, . B, Mn concentration-activity profiles. The enzymatic activity in COS-7 cells transfected with pcDNAI-mFuc-TIV or pcDNAI-hFuc-TIV was assayed using 20 mMN-acetyllactosamine and 3 µM GDP-[C]fucose (``Experimental Procedures''). Reactions contained 50 mM Tris-HCl, pH 7.2.

Figure 4: Apparent Michaelis constants for GDP-[C]fucose determined for mouse and human Fuc-TIVs. Apparent K values were determined (see ``Experimental Procedures'') in the presence of 20 mMN-acetyllactosamine, 15 mM Mn, and 50 mM Tris-HCl, pH 7.2. Cell extracts prepared from COS-7 cells transfected with pcDNAI-mFuc-TIV exhibited an apparent K of 16.6 µM, whereas human Fuc-TIV extracts generated using plasmid pcDNAI-hFuc-TIV maintained an apparent K of 27.0 µM.

Figure 5: Apparent Michaelis constants for N-acetyllactosamine and -(2-3)-sialyl-N-acetyllactosamine, determined for murine and human Fuc-TIVs. Apparent K values were determined (see ``Experimental Procedures'') in the presence of 3 µM GDP-fucose, 15 mM Mn, and 50 mM Tris-HCl, pH 7.2. A, N-acetyllactosamine K. Using N-acetyllactosamine as the acceptor, cell extracts prepared from COS-7 cells transfected with pcDNAI-mFuc-TIV exhibited an apparent K of 2.05 mM, whereas human Fuc-TIV extracts generated using plasmid pcDNAI-hFuc-TIV exhibited an apparent K of 3.82 mM. B, 2`-sialyl-N-acetyllactosamine K. Apparent K values using -(2-3)-sialyl-N-acetyllactosamine as the acceptor were 1.78 mM and 6.74 mM for the murine and human Fuc-TIVs, respectively. C, Gal1-3Gal1-4GlcNAc K. The apparent K value using Gal1-3Gal1-4GlcNAc as the acceptor was 0.71 mM for murine Fuc-TIV/ELFT.

Reactions containing neutral acceptors (N-acetyllactosamine, lactose, lacto-N-biose I, 2`-fucosyllactose, Gal1-3Gal1-4GlcNAc) were terminated by the addition of 20 µl of ethanol and 560 µl of water. Samples were centrifuged at 15,000 g for 5 min, and a 50-µl aliquot was subjected to scintillation counting to determine the total amount of radioactivity in the reaction. An aliquot of 200 µl was applied to a column containing 400 µl of Dowex 1-X2-400, formate form(35, 37) . The column was washed with 2 ml of water, and the radioactive reaction product, not retained by the column, was quantitated by scintillation counting. Reactions with the acceptor -(2-3)-sialyl-N-acetyllactosamine were terminated by adding 980 µl of 5.0 mM sodium phosphate buffer, pH 6.8. Samples were then centrifuged at 15,000 g for 5 min, and a 500-µl aliquot was applied onto a Dowex 1-X8-200 column (1 ml) prepared in the phosphate form. The reaction product was collected in the eluate and quantitated as described previously(43) .

The structure of the product obtained with -(2-3)-sialyl-N-acetyllactosamine was confirmed by HPLC fractionation, before and after neuraminidase digestion, as described previously(20) . The structures of the products obtained with the neutral acceptor substrates (N-acetyllactosamine, lactose, lacto-N-biose I, 2`-fucosyllactose) were also confirmed by HPLC fractionation, using methods described previously(20, 37, 44, 45) . The structure of the radiolabeled product (Gal1-3Gal1-4([C]Fuc1-3)GlcNAc) obtained with the neutral trisaccharide Gal1-3Gal1-4GlcNAc was confirmed in separate experiments, using methods described previously(20, 37) . The product was purified by chromatography on Dowex 1-X2-400, formate form(35, 37) , and subsequently fractionated by amine absorption HPLC (Dynamax 60A column, Rainin Instruments; isocratic gradient in 70% acetonitrile, 30% water; flow rate of 1 ml/min)(37, 44, 45) . This product was identified as a tetrasaccharide by virtue of co-elution, at 19 min, with a radiolabeled trisaccharide standard (Fuc(1-2)Gal1-4([C]Fuc1-3)GlcNAc). The tetrasaccharide product was digested with 0.01 units of jack bean -galactosidase (Boehringer Mannheim) for 1 h at 37 °C in 100 mM Tris, pH 6.5. The digest was desalted by Dowex chromatography and fractionated by HPLC exactly as described above for the product. The product of this digestion was identified as a trisaccharide by virtue of co-elution, at 13.4 min, with the trisaccharide standard (Gal1-4([C]Fuc1-3)GlcNAc.

In Situ Hybridization Analysis

Cryosections (10 µm thick) were prepared with Jung Frigocut 2800N cryostat (Leica) from organs of 129/J strain mice. Tissue sections were fixed in 4% paraformaldehyde/phosphate-buffered saline for 30 min at room temperature. After proteinase K treatment and then acetylation by acetic anhydride, sections were incubated with antisense or sense probes (2 10 cpm/µl) for 18 h at 55 °C in 20 mM Tris-HCL, pH 8.0, 300 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 50% formamide, 10% dextran sulfate, 5 Denhardt's solution, 50 mg/ml heparin, 0.1 M dithiothreitol, and 0.5 mg/ml Escherichia coli tRNA. The slides were washed for 30 min at 65 °C in 50% formamide, 2 SSC, 10 mM dithiothreitol. The sections were then subjected to digestion with 10 µg/ml of ribonuclease A at 37 °C to eliminate residual nonbase-paired probe and were then washed again in the wash solution described above. Slides were exposed for 2 weeks using Kodak NBT-2 emulsion and processed using D-19 developer and fixer (Eastman Kodak Co.). The sections were subsequently stained with hematoxylin and eosin.

Interspecific Mouse Backcross Mapping

A description of the probes and RFLPs for the loci linked to Fut4 including murine macrophage metalloelastase (Mmel), low density lipoprotein receptor (Ldlr), and erythropoietin receptor (Epor) has been reported previously(48) . Recombination distances were calculated as described previously (49) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

RESULTS

Molecular Cloning of a Murine 1-3FT Gene

This hybridization screen also identified phages containing a 1.4-kb NcoI-SspI fragment that cross-hybridizes with the human 1-3FT gene encoding Fuc-TIV(19, 20, 21) . Sequence analysis of the gene fragment identifies a single long open reading frame (Fig. 1), which begins with a methionine codon located within a sequence context largely consistent with Kozak's consensus rules for mammalian translation initiation(52) . Hydropathy analysis (53) of the protein sequence predicted by this open reading frame identifies a single 22-amino acid hydrophobic segment at the NH terminus, implying that the polypeptide has a type II transmembrane topology typical of mammalian glycosyltransferases(16, 54) . Sequence comparisons made between the predicted murine protein and human 1-3FTs identify significant primary sequence similarities; the murine protein maintains approximately 33, 33, 39, and 37% amino acid sequence identity with the human Fuc-TIII(37) , Fuc-TVI(45) , Fuc-TVII (55, 56) , and Fuc-TV (57) enzymes, respectively (data not shown). However, it is most similar to human Fuc-TIV(19, 20, 21) , which shares 77% amino acid sequence identity with the murine protein (304 identities at 396 aligned residues; Fig. 1). This sequence similarity includes the conservation of two consensus sites for asparagine-linked glycosylation (Fig. 1).

Maximal sequence similarity is achieved by aligning the murine DNA sequence in a colinear manner with the sequence of the human Fuc-TIV gene (19, 20) and its cDNA(21) . Like the human Fuc-TIV/ELFT locus, the murine gene apparently maintains a single coding exon. Primer extension experiments and RNase protection analyses designed to define the transcriptional initiation site for this murine gene have failed because of the tendency for its transcript to form secondary structure that is resistant to denaturation (data not shown). This is most probably a function, in part, of the extraordinarily high G+C content within the 5` end of this gene. We also believe these observations explain our inability to isolate full-length murine Fuc-TIV cDNAs from multiply screened high quality cDNA libraries (data not shown). Nonetheless, comparison of the DNA sequences of the murine and human genes through positions exceeding 400 base pairs proximal to their respective initiation codons discloses that their respective 5`-flanking regions are identical at 66% of the aligned positions (Fig. 1). This high level of DNA sequence identity is maintained throughout the region of the human gene where it is co-linear with Fuc-TIV/ELFT mRNA transcripts (Fig. 1), as defined by cDNA cloning experiments(21) , which in turn yield Fuc-TIV/ELFT or a longer form of this enzyme (ELFT-L; (21) ). The murine gene also has the potential to yield a longer form of its polypeptide product. This longer sequence is represented by a polypeptide initiating at a methionine codon located 33 codons 5` to the human Fuc-TIV/ELFT initiator methionine codon, extending the shorter murine polypeptide by 33 amino acid residues at its NH terminus (Fig. 1). This longer murine sequence maintains 52% amino acid sequence identity with the ELFT-L polypeptide in a region immediately proximal to the Fuc-TIV/ELFT initiator methionine residue (Fig. 1). These observations further support the conclusion that the murine gene maintains a structural organization essentially identical to that of the human Fuc-TIV/ELFT gene.

Expression of the Mouse 1-3FT Gene in COS-7 Cells

Figure 2: Flow cytometry histograms of COS-7 cells transfected with the murine Fuc-TIV gene. Cells transfected with either the 1-3FT expression vector pcDNAI-mFuc-TIV or the control vector pcDNAI were stained with the monoclonal antibodies indicated in the inset and were subjected to flow cytometry analysis (``Experimental Procedures''). The data presented are the mean (linear) fluorescence intensities of the antigen-positive population of transfected cells (see ``Experimental Procedures'').

In Vitro Enzyme Assay of Mouse and Human Fuc-TIVs

Kinetic analyses of murine and human Fuc-TIV enzymes indicate that they exhibit typical substrate concentration-dependent Michaelis-Menten kinetics ( Fig. 4and 5). The calculated apparent Michaelis constants for the donor substrate GDP-fucose are 16.6 and 27.0 µM for the murine and human enzymes, respectively (Fig. 4). The apparent Michaelis constants for the acceptor substrates N-acetyllactosamine and -(2-3)-sialyl-N-acetyllactosamine are 2.05 and 1.78 mM, respectively, for the murine enzyme, and 3.3 mM and 6.74 mM, respectively, for human Fuc-TIV (Fig. 5). Both enzymes utilize the neutral type II acceptor molecules 2`-fucosyllactose and lactose with efficiencies that are substantially less than those obtained with either -(2-3)-sialyl-N-acetyllactosamine or N-acetyllactosamine. For example, a single preparation of the murine enzyme utilized 2`-fucosyllactose and lactose at rates that were 7% (1.1 nmol/mgh) and 1% (0.2 nmol/mgh), respectively, of the rate obtained with N-acetyllactosamine (16.6 nmol/mgh). Only trace amounts of fucosylated product were produced when the neutral type I acceptor lacto-N-biose I was assayed with the same preparation of murine enzyme (0.05 nmol/mgh). These results are virtually identical to those obtained when extracts containing human Fuc-TIV activity are assayed with the latter three substrates(20, 55) . Thus, when considered together with the DNA and protein sequence comparisons and the flow cytometry analyses, these biochemical data indicate that the open reading frame shown in Fig. 1encodes an 1-3FT activity that corresponds to a human ``myeloid''-type 1-3FT (Fuc-TIV/ELFT)(19, 20, 21, 58, 59) .

The Gal1-3Gal1-4GlcNAc trisaccharide is a cell surface oligosaccharide epitope expressed by many murine tissues(60) . There is indirect evidence for a fucosylated form of this epitope in murine tissues (Gal1-3Gal1-4[Fuc1-3]GlcNAc; (10) ). In vitro studies using a human 1-3FT suggest that fucosylation can be a terminal step in the synthesis of this tetrasaccharide(61) . We find that the murine 1-3FT can efficiently utilize the trisaccharide Gal1-3Gal1-4GlcNAc (with an apparent K = 0.71 mM; Fig. 5C) to form a fucosylated product corresponding to the tetrasaccharide Gal1-3Gal1-4[Fuc1-3]GlcNAc (see ``Experimental Procedures''). This observation suggests that murine glycoconjugates containing the Gal1-3Gal1-4GlcNAc trisaccharide represent one authentic acceptor substrate for this murine 1-3FT. Although the human Fuc-TIV enzyme can also form this tetrasaccharide product in vitro (data not shown), this reaction has no apparent physiological relevance since the human genome does not encode a functional (1-3)-galactosyltransferase capable of synthesizing Gal1-3Gal1-4GlcNAc precursors(62).

Chromosomal Location of the Murine 1-3FT Locus

Figure 6: Fut4 maps in the proximal region of mouse chromosome 9. Fut4 was placed on mouse chromosome 9 by interspecific backcross analysis. The segregation patterns of Fut4 and flanking genes in 138 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 138 animals were typed (see text for details). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J M. spretus)F parent. The shadedboxes represent the presence of a C57BL/6J allele, and whiteboxes represent the presence of M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 9 linkage map showing the location of Fut4 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

The proximal region of mouse chromosome 9 shares a region of homology with human chromosomes 11q and 19p (summarized in Fig. 6). The human Fuc-TIV gene (locus designation FUT4) was initially assigned to human 11q12-qter(63) . More recently, the human map position has been refined to 11q21(64) . These studies provide additional support for concluding that this murine gene is the human homologue of Fuc-TIV gene, and confirm and extend the region of homology between mouse chromosome 9 and the long arm of human chromosome 11.

Tissue-specific Expression of the Murine Fuc-TIV Gene

Figure 7: Tissue-specific expression patterns of the murine Fuc-TIV gene. Polyadenylated RNA samples (3 µg) prepared from various murine tissues (panelA) and cell lines (panelB) were subjected to Northern blot analysis as described under ``Experimental Procedures.'' The blot was probed with the NcoI-SspI fragment using hybridization and wash conditions detailed under ``Experimental Procedures.'' Cell lines represent the following lineages: MEL, murine erythroleukemia cell line; P388 and RAW (RAW 264.7), macrophage; EL4, T-cell; S107, 63 (TH2.54.63), and 180.1, B-cell lines (hybridomas). RNA molecular size standards, in kb, are indicated at the left.

Northern blot analysis of cultured blood cell-type cell lines indicates that the Fuc-TIV transcript is relatively abundant in the murine erythroleukemia cell line MEL(26, 27) , substantially less abundant in the RAW macrophage-derived line(28, 29) , and not detectable in the P388 macrophage-derived cell line (30) or the three lymphoid cell lines we examined (T-cell line EL4, (23) ; mature B-cell lines (hybridomas) S107, (22) ; TH2.54.63, (24) ; and 180.1, (25) ). These results suggest that the murine Fuc-TIV gene, like its human counterpart, may be expressed in cells derived from the myeloid lineage but not in abundance in cells of the lymphoid lineage.

In situ hybridization analysis was used to delineate the cell types within some of the organs where relatively abundant levels of this transcript are present (Fig. 8). These experiments demonstrate that the Fuc-TIV transcript accumulates to substantial levels within the epithelial cells lining the stomach and colon (Fig. 8). The Fuc-TIV transcript accumulates to a lesser degree within the epithelial cells (both absorptive cells and goblet cells) lining the small intestinal villi, and the mucus glands within the small intestine. Fuc-TIV transcripts are also evident in the epithelial cells lining the epididymus (Fig. 8) but are not visible within the testis proper (data not shown). These observations correspond well with Fuc-TIV transcript abundance seen on the Northern blot analyses (Fig. 7). No Fuc-TIV transcripts are detected in the kidney or the lung by in situ hybridization (data not shown), even though low to moderate levels of the Fuc-TIV transcript are evident on Northern blot analysis. It remains to be determined if this apparent discrepancy may be accounted for by the relatively insensitive nature of the in situ hybridization method and/or by Fuc-TIV transcripts in blood cells within the larger vessels of these organs. These cells may contribute to the Northern blot signals but may be eliminated from the tissues prior to or during preparation for in situ hybridization. Fuc-TIV transcripts are not detected in murine neutrophils (data not shown); we have yet to define which blood cell lineages, and which maturation stage(s) of those lineages, are responsible for the Fuc-TIV transcripts observed in bone marrow mRNA.

Figure 8: In situ hybridization analysis of Fuc-TIV transcripts in murine tissues. Mouse stomach (A-C), small intestine (D-F), colon (G-I), and epididymus (J-L) were hybridized with antisense (A, B, D, E, G, H, J, K) or sense (C, F, I, L) mouse Fuc-TIV probes. PanelsA, D, G, and J are bright-field photomicrographs, and panels B, C, E, F, H, and I are dark-field photomicrographs, each taken at 40 magnification. MS, muscle layer; GL, glands; VL, villi. Arrows in panel J point to the tubular lumenae within the epididymus.

DISCUSSION

Biochemical studies indicate that selectin ligand expression in myeloid and lymphoid lineages is controlled in part by cell type-specific expression of one or more 1-3FTs and that these enzymes consequently play a pivotal role in the human inflammatory response(5, 6, 7, 8) . Evidence also suggests that the aberrant expression of these enzymes in malignancy may facilitate the spread of transformed cells via selectin-dependent metastatic processes(5, 6, 7, 63, 64, 65, 66, 67) . The developmentally regulated expression patterns of -(1-3)-fucosylated oligosaccharides in mammalian embryos(2, 9, 10, 11, 12) further imply that these molecules have additional, undefined functions. Given logistical and ethical considerations, the laboratory mouse is a useful system to analyze embryonic and adult 1-3FT gene expression patterns and to perturb these patterns through transgenic and embryonic stem cell approaches.

The polypeptide product of the murine gene described here shares 77% amino acid sequence identity with human Fuc-TIV(19, 20, 21) . Inter-species sequence comparisons have not previously been made for any 1-3FT sequences; the degree of sequence identity observed is comparable with levels reported for other cross-species comparisons of glycosyltransferase sequences (68, 69, 70) and suggest that this murine gene is the orthologous homologue (71) of human Fuc-TIV. This assignment is further supported by their corresponding chromosomal localizations and by the genomic organization of the murine gene and the human Fuc-TIV gene, both of which apparently maintain intronless coding sequences.

Comparison of the catalytic properties of the murine enzyme with those of the human Fuc-TIV enzyme provides additional evidence for their homologous nature. Transfection studies indicate that both enzymes can determine expression of surface-localized Lewis x molecules, but not sialyl Lewis x moieties or products based on type I precursors. The mouse and human enzymes maintain similar affinities for the type II acceptor N-acetyllactosamine, in vitro (2.05 and 3.3 mM, respectively), but differ somewhat in their apparent affinities for -(2-3)-sialyl-N-acetyllactosamine. Specifically, the human enzyme exhibits a rather higher apparent K (6.7 mM) for the sialylated substrate than does the mouse enzyme (1.8 mM). Nevertheless, the murine enzyme does not utilize oligosaccharides terminating with -(2-3)-sialylated type II chains when it is expressed in the cultured cell lines used here. The apparent discrepancy presented by the ability of Fuc-TIV to utilize -(2-3)-sialylatedtype II oligosaccharides in vitro, but not in vivo, may be resolved by considering recent results indicating that Fuc-TIV resides in a Golgi compartment proximal to -(2-3)-sialyltransferase and thus may not have an opportunity to operate upon such substrates within a cell(72) . These observations further emphasize that it is difficult to reliably predict the spectrum of oligosaccharide products that will be constructed in a specific cell lineage by a given glycosyltransferase solely by considering the results obtained from in vitro assays using low molecular weight oligosaccharide acceptors. This notion is strongly reinforced by results indicating that the human Fuc-TIV gene can, under some circumstances, determine cell surface sialyl Lewis x expression in transfected cells, and that this outcome is critically dependent upon the glycosylation phenotype of the host cell in which Fuc-TIV is expressed(73) . Thus, while it seems likely that the murine Fuc-TIV enzyme creates Fuc1-3GlcNAc linkages in murine tissues, it is not yet possible to predict which murine glycoconjugate acceptors will be utilized by this 1-3FT, and, consequently, we cannot yet predict the cell surface products created by this enzyme.

However, one such molecule may correspond to the terminal oligosaccharide structure Gal1-3Gal1-4GlcNAc. This determinant is widely expressed in murine tissues(60) , and, as we have shown here, is efficiently utilized, in vitro, by the murine Fuc-TIV/ELFT enzyme. Prior work has provided indirect evidence for the expression of the -(1-3)-fucosylated form of this molecule (Gal1-3Gal1-4[Fuc1-3]GlcNAc) by murine cell surface glycoconjugates during the periimplantation period(10) . This tetrasaccharide has also been synthesized in vitro by a sequential enzyme-assisted synthetic scheme involving -(1-3)-galactosylation of N-acetyllactosamine to form Gal1-3Gal1-4GlcNAc, followed by -(1-3)-fucosylation with an 1-3FT isolated from human milk(61) . However, since in some cell types the murine 1-3 galactosyltransferase responsible for synthesis of the Gal1-3Gal1-4GlcNAc precursor trisaccharide is most probably localized to a Golgi compartment coincident with -(2-3)-sialyltransferase, and distal to the compartment where Fuc-TIV is located(74) , it seems probable that simultaneous expression of the Fuc-TIV and -(1-3)-galactosyltransferase genes will not necessarily yield expression of this tetrasaccharide. Additional information concerning the expression pattern of this tetrasaccharide will await the development of antibodies directed against this molecule.

The Gal1-3Gal1-4[Fuc1-3]GlcNAc tetrasaccharide represents a noncharged analogue of the sialyl-Lex determinant, prompting a suggestion that it may represent a possible ligand for the selectins (61) . It is not yet known if this tetrasaccharide can participate in selectin-dependent cell adhesion processes or if it is expressed by murine neutrophils, although the precursor trisaccharide determinant is displayed by these cells. ()It is interesting to note in this context that the oligosaccharide portion of the murine E- and P-selectin counter-receptors are not yet defined, although the sialyl Lewis x determinant is not expressed on murine leukocytes(75) , at least as defined by the use of the monoclonal antibody CSLEX. The tissue-specific expression patterns, and functions, if any, of this tetrasaccharide molecule thus remain an open and interesting question.

Northern blot analyses identify a single mouse Fuc-TIV transcript in all tissues where the gene is expressed, whereas the human Fuc-TIV gene generates multiple transcripts(19, 20, 21) . By analogy to other mammalian glycosyltransferase mRNAs(16, 54) , the relatively large size of the mouse Fuc-TIV transcript suggests that it contains substantial amounts of 3`- and/or 5`-untranslated segments. The structure of this transcript remains to be determined by cloning and sequencing of the cDNA(s) derived from this gene. While the murine transcript is detectable in bone marrow, the specific marrow cell types that express this gene remain to be precisely defined. It is likely, however, that the marrow transcripts are derived in part from myeloid-lineage cells since the human Fuc-TIV gene is expressed in myeloid cells(19, 20, 21) . The murine erythroid lineage may also contribute to expression in the bone marrow since transcripts are detected in the mouse erythroleukemia line MEL. The murine transcript is also relatively abundant in the epithelial cells lining the colon and stomach, with somewhat lesser amounts in small intestinal epithelial cells. The function of this enzyme in these locations remains unknown, although it is interesting to speculate that it serves to participate in the synthesis of fucosylated mucins that may operate to protect the gastrointestinal lining from destructive effects of digestive enzymes or from ingested pathogens. The low levels of the Fuc-TIV transcript in the testes is accounted for by transcripts that accumulate in the epididymus. The low levels of Fuc-TIV message detected in the kidney, and the trace amounts identified in brain and heart, may represent transcripts derived from specific cell types within the parenchyma of these organs. Further in situ hybridization experiments will be required to confirm this possibility.

These results indicate that while the murine and human Fuc-TIV genes are similarly organized, there may be substantial interspecies differences in the structural, functional, and regulatory properties of the orthologous 1-3FTs. Evidence suggests that the functional roles of their oligosaccharide products may also differ in significant ways. The expression patterns exhibited by the mouse 1-3FT gene are different from those shown by a rat -(2-6)-sialyltransferase gene (76, 77) , a constitutively expressed mouse -(1-4)-galactosyltransferase gene(78, 79) , a mouse -(1-3)-galactosyltransferase gene(60) , and a series of human sialyltransferase genes. When considered together with these data, our results provide additional support for the notion that transcriptional control of glycosyltransferase gene expression regulates cell surface glycosylation(54) .

Functional correlates for the regulation of this 1-3FT remain to be explored. In this context, we have compared our interspecific map of chromosome 9 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations. ()Fut4 maps in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). The task of assigning function to this locus may be facilitated by a more complete understanding of the types, structures, and expression patterns of this and other murine glycosyltransferase genes and their cognate oligosaccharide products, in concert with approaches that utilize transgenic and gene ablation technologies in the mouse.

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant 1R01GM47455 (to J. B. L.) and by the National Cancer Institute, under contract NO1-CO-74101 with ABL (to N. A. J., and N. E. C). Computer-based DNA sequence analysis was supported in part by National Institutes of Health Grant M01RR00042 to the General Clinical Research Center at the University of Michigan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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(TM)/EMBL Data Bank with accession number(s) U33457 [GenBank]and U33458[GenBank].

§

Partially supported by the National Institutes of Health Predoctoral Training Grant 5T32GM07315 to the Cell and Molecular Biology Graduate Program at the University of Michigan.

¶

An Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., Medical Science Research Bldg. I, Rm. 3510, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650. Tel.: 313-747-4779; Fax: 313-936-1400.

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The abbreviations used are: 1-3FT, -(1-3)-fucosyltransferase, ELFT, ELAM-1 ligand fucosyl transferase; FCS, fetal calf serum; MEL, murine erythroleukemia cell line; HPLC, high performance liquid chromatography; bp, base pair(s); kb, kilobase(s); GDP-fucose, -D-N-acetylglucosaminide 3--L-fucosyltransferase.

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A. Thall and J. B. Lowe, unpublished data.

()

Compiled by M. T. Davisson, T. H. Roderick, A. L. Hillyard, and D. P. Doolittle and provided from GBASE, a computerized data base maintained at the Jackson Laboratory, Bar Harbor, ME.

ACKNOWLEDGEMENTS

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