Peter L. Smith

The α(1.3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis.

alpha(1,3)Fucosylated oligosaccharides represent components of leukocyte counterreceptors for E- and P-selectins and of L-selectin ligands expressed by lymph node high endothelial venules (HEV). The identity of the alpha(1,3)fucosyltransferase(s) required for their expression has been uncertain, as has a requirement for alpha(1,3)fucosylation in HEV L-selectin ligand activity. We demonstrate here that mice deficient in alpha(1,3) fucosyltransferase Fuc-TVII exhibit a leukocyte adhesion deficiency characterized by absent leukocyte E- and P-selectin ligand activity and deficient HEV L-selectin ligand activity. Selectin ligand deficiency is distinguished by blood leukocytosis, impaired leukocyte extravasation in inflammation, and faulty lymphocyte homing. These observations demonstrate an essential role for Fuc-TVII in E-, P-, and L-selectin ligand biosynthesis and imply that this locus can control leukocyte trafficking in health and disease.

The α(1,3)fucosyltransferases FucT-IV and FucT-VII Exert Collaborative Control over Selectin-Dependent Leukocyte Recruitment and Lymphocyte Homing

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Mvwf, a Dominant Modifier of Murine von Willebrand Factor, Results from Altered Lineage-Specific Expression of a Glycosyltransferase

We have identified altered lineage-specific expression of an N-acetylgalactosaminyltransferase gene, Galgt2, as the gain-of-function mechanism responsible for the action of the Mvwf locus, a major modifier of plasma von Willebrand factor (VWF) level in RIIIS/J mice. A switch of Galgt2 gene expression from intestinal epithelial cell-specific to a pattern restricted to the vascular endothelial cell bed leads to aberrant posttranslational modification and rapid clearance of VWF from plasma. Transgenic expression of Galgt2 directed to vascular endothelial cells reproduces the low VWF phenotype, confirming this switch in lineage-specific gene expression as the likely molecular mechanism for Mvwf. These findings identify alterations in glycosyltransferase function as a potential general mechanism for the genetic modification of plasma protein levels.

Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus

Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects. We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP–fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype. Live-born FX(−/−) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet. FX(−/−) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc. Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP–fucose synthesis. Conditional control of fucosylation in FX(−/−) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion.

Molecular Cloning and Expression of GDP-d-mannose-4,6-dehydratase, a Key Enzyme for Fucose Metabolism Defective in Lec13 Cells

Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns. The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes. Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans. However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian α-1,3-fucosyltransferase and α-1,2-fucosyltransferase gene families. To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-d-mannose-4,6-dehydratase (GMD). This enzyme generates GDP-mannose-4-keto-6-d-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-d-deoxymannose epimerase/GDP-4-keto-6-l-galactose reductase) to GDP-l-fucose. GMD is thus imperative for the synthesis of all fucosylated oligosaccharides. An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs. This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to anArabidopsis thaliana GMD cDNA. This approach, together with 5′-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line. Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells. By contrast, the transcript encoding the FX protein, which forms GDP-l-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells. These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein. The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.

Detection of N-acetylgalactosaminyltransferase mRNA which determines expression of Sda blood group carbohydrate structure in human gastrointestinal mucosa and cancer

The Sda blood group carbohydrate structure, GaINAcβl‐4[NeuAcα2–3]Galβl‐4GlcNAc‐R, is expressed on glycolipid and glycoprotein in human gastrointestinal mucosa. The expression of the Sda determinant dramatically decreases in cancer tissue. The activity of the βl,4N‐acetylgalactosaminyltransferase (Sda‐GaINAcT), which transfers GaINAc to NeuAcα2–3Galβl‐4Glc(NAc)‐R, correlates with the expression of the Sda immuno‐epitope. From the total RNA fraction of human gastric mucosa, we have amplified a cDNA segment by reverse‐transcription‐polymerase‐chain reaction (RT‐PCR), using primers designed according to the cDNA sequence of a murine βl,4GalNAcT which synthesizes the Sda determinant. An RT‐PCR product of 390 bp shared 85% nucleotide identity with the murine Sda‐related βl,4GalNAcT. This RT‐PCR product hybridized to a transcript in mRNA prepared from human gastric mucosa. In RT‐PCR using specific primers to this PCR product, Sda‐GaINAcT mRNA was detected in all samples of normal stomach and small intestine examined and the majority of normal colonic specimens. Six out of nine cases of gastric cancer, and 9 out of 13 cases of colonic cancer failed to produce the target DNA. These results correlate with the βl,4GalNAcT activity measured in the same samples. In conclusion, a segment of the cDNA for βl,4GalNAcT which determines expression of the Sda carbohydrate structure was obtained, and reduced transcription of this βl,4GalNAcT resulted in the disappearance of the Sda epitope in gastrointestinal cancer.

Strain-specific modification of lethality in fucose-deficient mice

The FX locus encodes an essential enzyme in the de novo pathway of GDP-fucose biosynthesis. Mice homozygous for a targeted mutation of the FX gene manifest a host of pleiotropic abnormalities including a lethal phenotype that is almost completely penetrant in heterozygous intercrosses on a mixed genetic background. Here we have investigated genetic suppression of FX-mediated lethality. Reduced recovery of heterozygous mice was observed while backcrossing the null FX allele to C57BL/6J (B6), but was less dramatic in an outcross to CASA/Rk and absent in an outcross to 129S1/SvImJ, indicating that genetic background modifies survival of FX+/− progeny. Substantial strain-specific differences in pre- and postnatal survival of FX−/− progeny were also detected in heterozygous crosses of C57BL/6J congenic, 129S1B6F1, and B6CASAF1 mice. Specifically, intrauterine survival of FX−/− mice was greatly increased during a heterozygous intercross on a uniform C57BL/6J genetic background compared with survival on a hybrid genetic background consisting of a mixture of C57BL/6J and 129S2/SvPas. In addition, statistically significant clustering of FX−/− progeny into litters and specific breeding cages was noted during a B6CASAF1 FX+/− intercross, suggesting a rare mechanism for modifier gene action in which parentally expressed genes define the phenotype, in this case the survival potential, of mutant offspring. Our results disclose that lethality in FX mutant mice is determined by one or more strain-specific modifier loci.

S2.1 Structure and function of mammalian glycosyltransferase genes

Purification of UDP-GalNAc: polypeptide N-acetylgalactosaminyl transferase from human placenta and ovine and porcine submaxillary glands by affinity chromatography on a defined synthetic peptide containing multiple threonine acceptor substrate sites resulted in separation of at least two distinct threonine transferase activities. A panel of synthetic peptides was utilized as acceptor substrates on transferase preparations before and after affinity purification on the synthetic peptide in order to evaluate the substrate specificities. Only a fraction of the transferase activity available was bound to the affinity column evaluated by a number of acceptor substrate peptides including the peptide used for the affinity chromatography column. Interestingly, one peptide containing a single threonine glycosylation site was not glycosylated by the affinity purified transferase and the transferase activity to this peptide passed quantatively through the affinity column. Furthermore, kinetic analysis of crude and purified transferase preparations revealed significant differences between the two transferase preparations. The results suggest that more than one threonine transferase activity exist, and it is likely to be due to different structural proteins.