Frances I. Smith

Cloning of a rat α1,3-fucosyltransferase gene: a member of the fucosyltransferase IV family

We report the cloning of a rat α1,3-fucosyltransferase gene (rFuc-T), isolated from a rat genomic library by a PCR-cross-hybridization based cloning approach using primers derived from the conserved region of human α1,3-Fuc-T sequences. Comparison of the rFuc-T predicted amino acid sequence with those of previously cloned human and murine fucosyltransferases showed highest degree of homology to murine Fuc-TIV (87% identity) and human Fuc-TIV (78% identity), with lower homology (41–49% identity) to Fuc-TIII, V, VI, and VII. COS-1 cells transfected with the rFuc-T gene expressed a fucosyltransferase activity with type 2 (Galβ1→4GlcNAc)-containing oligosaccharides and the glycolipid acceptor neolactotetraosyl-ceramide but only low activity with sialylated substrates; the SSEA-1/Lex antigen was detected in transfected cells by immunocytochemistry. Based on these results, we surmise that rFuc-T is a member of the fucosyltransferase IV family. Northern blot analysis with a rFuc-T specific probe indicated a major transcript of 4.2 kb most abundantly expressed in rat spleen; minor transcripts of different sizes were detected in several tissues, including rat brain. Abbreviations: Fuc-T, fucosyltransferase; SSEA-1, stage specific embryonic antigen or Lewisx (Lex); ELFT, ELAM-1 ligand fucosyltransferase; ELAM, endothelial-leukocyte adhesion molecule; nLc4Cer, neolactotetraosylceramide (Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→1Cer); NEM, N-ethylmaleimide

N-Glycosylation site occupancy of rat α-1,3-fucosyltransferase IV and the effect of glycosylation on enzymatic activity

All mammalian alpha-1,3-fucosyltransferases (Fuc-Ts) so far characterized have potential N-glycosylation sites, but the role of these sites in enzymatic activity or localization has not been investigated. When one member of this family, rFuc-TIV, is expressed in bacteria, the unglycosylated form of rFuc-TIV has no detectable enzymatic activity. The two potential N-glycosylation sites of rFuc-TIV were mutated to determine site occupancy and the effect of site occupancy on enzyme activity and targeting of this enzyme. Results obtained with singly mutated forms of rFuc-TIV indicate that both sites are occupied in mammalian cells. Lack of glycosylation at sites 117-119, 218-220, or both of these sites, decreased enzyme activity to approximately 64%, 5% or 1%, respectively, of that seen in the unmutated enzyme. These results show that N-glycosylation is necessary for optimal enzyme activity, with glycosylation at site 218-220 playing the major role. However, N-glycosylation does not appear to affect the major intracellular location of the enzyme, as immunocytochemistry reveals the same perinuclear pattern of staining for the unglycosylated mutants as is seen for the wild-type rFuc-TIV in transfected cells.

α1,3 Fucosyltransferase, α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, and Lex glycoconjugates in developing rat brain

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Lex) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15–P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10–P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an α1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5–10) with different types of acceptors, and the differential expression of Lex containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, α-L-fucosidase, β-D-galactosidase and α-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

Regulation of the human acid β-glucosidase promoter in multiple cell types

Abstract Acid β-glucosidase (βGlc) is a housekeeping enzyme whose expression is ubiquitous, but differs greatly according to tissue of origin. Expression of a reporter gene under the control of a 622 bp fragment of the βGlc promoter correlated roughly with the relative amount of βGIc mRNA detected in five different cell lines, suggesting that elements within this region play a role in determining differential expression of the βGlc gene. Experiments using deletion mutants revealed that differential expression of βGlc is not due to the presence of promoter elements that are active in only certain cell types, but rather due to subtle changes in the magnitude of the effect of the different elements. Strikingly, regulatory elements located upstream of the TATA box are dispensible in several cell types, whereas elements located within exon 1 of the βGlc gene are essential for reporter gene expression in cultured cells. At least two exon 1 elements regulate mRNA levels, and one double stranded probe containing exon 1 sequences binds a factor present in extracts from HeLa and glioblastoma cells. Additionally, at least two of the exon 1 elements act in an orientation-independent fashion. Thus, it is likely that at least a subset of the exon 1 elements act as transcriptional enhancers.

Molecular cloning of rat ?1,3-fucosyltransferase IX (Fuc-TIX) and comparison of the expression of Fuc-TIV and Fuc-TIX genes during rat postnatal cerebellum development

Fucosylated glycoconjugates play an essential role in central nervous system development, but the regulation of expression of these molecules is not well understood. The final biosynthetic step for a major group of cerebellar fucosylated glycoconjugates (those bearing the developmentally regulated epitope 3‐fucosyl‐N‐acetyllactosamine, CD15, and related fucosylated epitopes) is catalyzed by an α‐1,3‐fucosyltransferase (FucT). The major FucT activity in postnatal rat cerebellum has a specificity consistent with that encoded by either a Fuc‐TIV‐ or Fuc‐TIX‐like gene, and thus the expression of these genes was investigated during postnatal rat cerebellar development. A rFuc‐TIX cDNA was cloned and a comparison of its enzymatic activity with rFuc‐TIV revealed similar results on oligosaccharides, but strikingly higher activity on lipid acceptors, suggesting a greater role for rFuc‐TIX than rFuc‐TIV in the synthesis of CD15 glycolipids. rFuc‐TIX mRNA levels were much higher than those of rFuc‐TIV in neonatal cerebellum. Whereas rFuc‐TIX mRNA levels remained relatively constant, rFuc‐TIV mRNA levels declined during postnatal cerebellar development. In situ hybridization of postnatal rat cerebella also revealed different patterns of expression for these two genes. The rFuc‐TIV gene was expressed predominantly in Purkinje cells and the deep cerebellar nuclei throughout postnatal development, but was expressed in granule neurons only in the neonatal, and not the adult, rat. In contrast, the rFuc‐TIX gene was expressed in cells in the granule cell layers in both neonatal and in the adult rat. The potential implications of the different enzymatic activities and cell localization of rFuc‐TIV and rFuc‐TIX expression for the regulation of fucosylated glycoconjugates during cerebellar development are discussed. J. Neurosci. Res. 62:206–215, 2000.

Regulation of expression of the gene encoding human acid β-glucosidase in different cell types

Acid beta-glucosidase (beta Glc) activity and mRNA levels were measured in several human cell lines, and found to vary over 50-fold. A comparison between relative levels of beta Glc enzyme and mRNA levels revealed three patterns. The first group, including epithelial, lymphoblast, histiocyte, glioblastoma and astrocytoma cell lines, showed a direct relationship between relative levels of mRNA and enzyme activity, indicating that mRNA levels play an important role in determining enzyme activity. The second group, including fibroblast, promyelocyte and neuroglioma cell lines, also showed a direct relationship between beta Glc enzyme and mRNA levels within this group, but had enzyme activities that were approximately sixfold higher than expected, when compared with enzymes within the first group. The third pattern was exhibited by a single monocyte cell line, which showed high levels of beta Glc mRNA, but only intermediate levels of enzyme activity. These results suggest that although beta Glc mRNA levels play a major role in regulating beta Glc activity, other mechanisms also influence enzyme levels in certain cell lines. These results also demonstrate the importance of examining several different cell types when considering mechanisms of housekeeping gene regulation. Additionally, culturing cells in the presence of the beta Glc-specific inhibitor, conduritol-B-epoxide, did not affect beta Glc mRNA levels, and cells derived from normals had levels of beta Glc mRNA comparable to those from Gaucher disease patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction of high CD15 expression to a subset of rat cerebellar astroglial cells can be overcome by transduction with adenoviral vectors expressing the rat α1,3-fucosyltransferase IV gene

Glycoconjugates bearing the epitope 3‐fucosyl‐N‐acetyllactosamine (CD15) are believed to be involved in cell‐cell interactions and are temporally and spatially regulated in the brain. In the rat postnatal cerebellum, CD15 is predominantly expressed in the molecular layer by Bergmann glial cells, but little CD15 expression is seen in other astroglia, and the basis for this restricted expression is not known. Adenoviral vectors were shown to efficiently deliver transgenes to cerebellar glial cells and were used to determine whether manipulation of glycosyltransferase activities could enhance the expression of CD15 in these cells. In dissociated cerebellar cell cultures, few glial cells normally express CD15. However, transduction of these cells with an adenoviral vector (AdGFPCMVFucT) that expressed both green fluorescent protein (GFP) and FLAG‐tagged rat α1,3‐fucosyltransferase IV (rFuc‐TIV) resulted in high CD15 expression on the surface of all transduced glial cells. Likewise, infection of cerebellar slice cultures caused the appearance of CD15‐positive transduced cells of glial cell morphology in the internal granule cell layer. Thus, enhancement of Fuc‐T activity caused robust CD15 expression in cerebellar glial cells that normally show little expression of CD15, suggesting a role for Fuc‐T levels in regulating CD15 expression in this cell type. The manipulation of levels of glycosyltransferases using adenoviral vectors may prove a useful tool to investigate questions of glycoconjugate regulation in glial cells in the developing rodent cerebellum. GLIA 31:144–154, 2000.

Viral vector‐mediated delivery of competing glycosyltransferases modifies epitope expression cell specifically

The glycoconjugate epitopes 3‐fucosyl‐N‐acetyllactosamine (CD15) and sulfoglucuronylcarbohydrate (SGC) mediate cell adhesion events in several systems, and are regulated both spatially and temporally during cerebellar development. In cotransfection studies using COS‐1 cells, competition between glycosyltransferases that utilize a common precursor involved in the final synthetic steps of these epitopes, can modulate epitope expression. For example, cotransfection of rat α1,3‐fucosyltransferase IV (Fuc‐TIV) and either rat glucuronic acid transferase P (GlcAT) or pig α1,3‐galactosyltransferase (GalT) resulted in the dominance of either SGC or GalαGal epitope expression, respectively, with blockage of CD15 epitope expression. Viral vectors expressing these glycosyltransferases were used to determine whether competition plays a role in establishing epitope dominance in cerebellar cells, and whether overexpression of competing glycosyltransferases could be used to block epitope expression. Infection of cerebellar astrocytes with viral vectors expressing either Fuc‐TIV, or Fuc‐TIX, caused dramatic increases in CD15 expression in the presence of continued endogenous SGC epitope expression. Likewise, viral transduction with GalT resulted in GalαGal expression without affecting endogenous CD15 or SGC expression. Thus, competition between these enzymes does not appear to play a role in establishing epitope expression in astrocytes, and transduction of these enzymes does not provide a method of blocking the expression of endogenous epitopes. In contrast to what was observed for astrocytes, infection with viral vectors expressing either Fuc‐T, GlcAT, or GalT did not result in significant expression of the relevant epitopes (CD15, SGC or GalαGal, respectively) on granule neurons. These results suggest a different complement of precursors are present in granule neurons and astrocytes, presumably due to the presence of different complements of glycosyltransferases in these cells.

The rat alpha1, 3-fucosyltransferase (rFucT-IV) gene encodes both long and short forms of the enzyme which share the same intracellular location.

Fucosyltransferase (FucT) activity has been detected on the surface of mouse germ cells and rat Sertoli cells, and has been postulated to play a role in cell-cell interactions. A recently cloned rat FucT (rFucT-IV) is expressed in the testes, and thus is a candidate for encoding the cell-surface FucT activity. This study maps the 5′-ends of several rFuc-T-IV mRNAs, and these results suggest that initiation of transcription may occur both upstream of the first ATG, as well as between the first two closely spaced, in-frame ATGs. Thus, in certain tissues, notably spleen, significant amounts of both a long and a short form of rFucT-IV would be predicted. This study also determines some basic properties of both the long and short forms of rFucT-IV, and investigates whether the use of alternative ATGs would allow FucT activity to be expressed both on the cell surface and in the Golgi. Plasmids that encode FLAG-epitope-labeled rFucT-IVs that initiate from either of the two ATGs were constructed, and rFucT-IV was expressed either in vitro using cell-free rabbit reticulocyte lysate, or after transfection in tissue culture. The results from these studies demonstrate that rFucT-IV is a glycosylated, transmembrane protein with a short cytoplasmic tail, and that either of the two ATGs in the 5′ region of the rFucT-IV gene are capable of acting as functional initiators of translation in vitro, to produce enzymatically active glycoproteins. However, no difference in the intracellular localization between the transferase containing a 48 amino acid or a 15 amino acid cytoplasmic tail was detected by immunocytochemistry, as both show the same pattern of Golgi-like staining in several different cell types, with no indication of surface expression. Thus, the additional amino-terminal 33 amino acids of the long form of rFucT-IV do not appear to influence its intracellular location in the cell types investigated.

Expression of myelin proteolipid protein in oligodendrocytes and transfected cells

The data presented in this paper show that the appropriate tools are now available to study the behavior of PLP and DM20 transcripts engineered with either point mutations or deletion of specific domains. Such studies should begin to provide new insights into the functions of PLP and DM20 and their role in relation to the optimal functioning of the nervous system.