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Myelin-associated glycoprotein (MAG, Siglec-4) is a quantita-tively minor membrane component expressed preferentially onthe innermost myelin wrap, adjacent to the axon. It stabilizesmyelin–axon interactions by binding to complementary ligandsontheaxolemma.MAG,amemberoftheSiglecfamilyofsialicacid–binding lectins, binds specifically to gangliosides GD1aand GT1b, which are the major sialoglycoconjugates onmammalian axons. Mice with a disrupted Galgt1 gene lackUDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase(GM2/GD2 synthase) and fail to express complex brain gang-liosides, including GD1a and GT1b, instead expressing acomparable amount of the simpler gangliosides GM3, GD3,and O-acetyl-GD3. Galgt1-null mice produce similar amountsoftotalmyelincomparedtowild-typemice,butasthemiceage,theyexhibitaxondegenerationanddysmyelinationwithaccom-panyingmotorbehavioraldeficits.HerewereportthatGalgt1-null mice display progressive and selective loss of MAG fromthebrain.At1.5monthsofage,MAGexpressionwassimilarinGalgt1-null and wild-type mice. However, by 6 months of ageMAG was decreased ~60% and at 12 months of age ~70% inGalgt1-nullmicecomparedtowild-typelittermates.Expressionof the major myelin proteins (myelin basic protein and proteo-lipid protein) was not reduced in Galgt1-null mice compared towild type. MAG mRNAexpressionwasthesamein12-month-old Galgt1-null compared to wild-type mice, an age at whichMAG protein expression was markedly reduced. We concludethat the maintenance of MAG protein levels depends on thepresence of complex gangliosides, perhaps due to enhancedstability when MAG on myelin binds to its complementaryligands, GD1a and GT1b, on the apposing axon surface.Key words: Galgt1/N-acetylgalactosaminyltransferase/GD1a/GT1b/MAGIntroductionMyelin-associated glycoprotein (MAG) is a minor myelincomponent, making up ~1% of central nervous systemand ~0.1% of peripheral nervous system myelin proteins(Trapp, 1990). It is preferentially expressed on the inner-most, periaxonal myelin membrane (Trapp et al., 1989),where it functions in myelin–axon interactions (Schachnerand Bartsch, 2000). The axon is dependent on signalsfrom myelin, specifically MAG, for its cytoarchitecture,structure, and long-term stability (Bjartmar et al., 1999).Genetic ablation of MAG results in reduced axon caliber,reduced axon neurofilament spacing and phosphoryla-tion, and progressive axon degeneration (Fruttiger et al.,1995; Li et al., 1994; Montag et al., 1994; Yin et al.,1998). These observations led to the conclusion thatMAG is an important signaling molecule in myelin–axon interactions and is required for optimal long-termaxon stability (Schachner and Bartsch, 2000). In addition,MAG inhibits axon regeneration after injury (Li et al.,1996; McKerracher et al., 1994; Mukhopadhyay et al.,1994). Along with Nogo, oligodendrocyte myelin glyco-protein, and chondroitin sulfate proteoglycans, MAGcontributes to a central nervous system environment thatis highly inhibitory for nerve regeneration (Fournier andStrittmatter, 2001; Sandvig et al., 2004; Wang et al., 2002).MAG, expressed on myelin, binds to complementaryligands on the apposing axon surface. MAG is a memberof the Siglec family of sialic acid–binding Ig-family memberlectins (Crocker and Varki, 2001; Crocker et al., 1996).Siglecs share significant domain and sequence similarityand bind to sialic acid–bearing glycoconjugates withvarying specificities for the sialic acid linkage and penulti-mate saccharides. Gangliosides are the major sialoglyco-conjugates in the brain (Schnaar, 2000) (Figure 1). MAGbinds with high affinity and specificity to two majorbrain gangliosides, GD1a and GT1b, that are expressedprominently on axons (DeVries and Zmachinski, 1980)and that bear the MAG-binding terminal sequenceNeuAca1-3Galb1-3GalNAc (Collins et al., 1997; Yanget al., 1996). Mice engineered to lack a key enzyme in gang-lioside biosynthesis, UDP-N-acetyl-D-galactosami-ne:GM3/GD3 N-acetyl-D-galactosaminyltransferase (EC2.4.1.92), do not express the NeuAca1-3Galb1-3GalNAc

The glycosyltransferases are recognized as a functional family of an estimated 300 distinct, intracellular, membrane-bound enzymes that are positioned along the secretory pathway and participate coordinately in the biosynthesis of the carbohydrate moieties on glycoconjugates. The full-length cDNA sequence for many of these proteins is now available yet little is known about the transcriptional or translational regulation of a given transcript or its decay rate in the cell. These issues are made more complex by the observations that transcription of a glycosyltransferase gene in different cells/tissues results in mRNAs with significantly different structures. For example, transcription of the murine beta4-galactosyoltransferase-1 gene in somatic cells yields two transcripts of 3.9 and 4.1 kb. In contrast transcription of this gene in developing male germ cells results in transcripts of 2.9 and 3.1 kb which are distinguished from their somatic cell counterparts primarily by the deletion of approximately 1.7 kb of sequence in the 3'-untranslated region (UTR). With the long range goal of determining the role that the 3'-UTR serves in mRNA decay we have taken advantage of a recently developed methodology, the Tet-Off system, to determine the half-life of the mRNA encoding beta4-galactosyltransferase-1 in the murine NIH 3T3 somatic cell line. We show that the beta4-galactosyltransferase-1 mRNA has a half-life of approximately 84 min (range of 82-85 min) in 3T3 cells and that substitution of the galactosyltransferase coding sequence with the coding sequence of luciferase does not significantly alter the decay rate (approximately 87 min; range of 84-91 min). This latter observation suggests that the beta4-galactosyltransferase-1 coding sequence does not contain functional elements that affect the intrinsic stability of this mRNA.

Six β4-galactosyltransferase (β4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat−2 line of Chinese hamster ovary cells (Gat−2 CHO). Two independent mutants termed Pro−5Lec20 and Gat−2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro−5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in β4GalT-1 transcripts in Pro−5Lec20 cells. The Gat−2Lec20 mutant expressed β4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the β4GalT-1 gene coding region. Northern analysis with probes from the remaining five β4GalT genes revealed that Gat−2 CHO and Gat−2Lec20 cells express all six β4GalT genes. Unexpectedly, the β4GalT-6 gene is not expressed in either Pro−5 or Pro−5Lec20 cells. Thus, in addition to a deficiency in β4GalT-1, Pro−5Lec20 cells lack β4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the β4GalT-1− (Gat−2Lec20) and β4GalT-1−/β4GalT-6−(Pro−5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennaryN-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, β4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. β4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivosubstrates of different β4GalTs. They may be used in glycosylation engineering and in investigating roles for β4GalT-1 and β4GalT-6 in generating specific glycan ligands.

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.

From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.

Beta1,4-galactosyltransferase (beta4GalT-I) is a constitutively expressed trans-Golgi enzyme, widely distributed in vertebrates, which synthesizes the beta4-N-acetyllactosamine structure commonly found in glycoconjugates. In mammals beta4GalT-I has been recruited for a second biosynthetic function, the production of lactose; this function takes place exclusively in the lactating mammary gland. In preparation for lactose biosynthesis, beta4GalT-I enzyme levels are increased significantly. We show that mammals have evolved a two-step mechanism to achieve this increase. In step one there is a switch to the use of a second transcriptional start site, regulated by a stronger, mammary gland-restricted promoter. The transcript produced is distinguished from its housekeeping counterpart by the absence of approximately 180 nt of 5'-untranslated sequence. In step two, this truncated transcript is translated more efficiently, relative to the major transcript expressed in all other somatic tissues.

Two distinct but related groups of cDNA clones, CKbeta4GT-I and CKbeta4GT-II, have been isolated by screening a chicken hepatoma cDNA library with a bovine beta1,4-galactosyltransferase (beta4GT) cDNA clone. CKbeta4GT-I is predicted to encode a type II transmembrane glycoprotein of 41 kDa with one consensus site for N-linked glycosylation. CKbeta4GT-II is predicted to encode a type II transmembrane glycoprotein of 43 kDa with five potential N-linked glycosylation sites. At the amino acid level, the coding regions of CKbeta4GT-I and CKbeta4GT-II are 52% identical to each other and 62 and 49% identical, respectively, to bovine beta4GT. Despite this divergence in amino acid sequence, high levels of expression of each cDNA in Trichoplusia ni insect cells demonstrate that both CKbeta4GT-I and CKbeta4GT-II encode an alpha-lactalbumin-responsive, UDP-galactose:N-acetylglucosamine beta4-galactosyltransferase. An analysis of CKbeta4GT-I and CKbeta4GT-II genomic clones established that the intron positions within the coding region are conserved when compared with each other, and these positions are identical to the mouse and human beta4GT genes. Thus CKbeta4GT-I and CKbeta4GT-II are the result of the duplication of an ancestral gene and subsequent divergence. CKbeta4GT-I maps to chicken chromosome Z in a region of conserved synteny with the centromeric region of mouse chromosome 4 and human chromosome 9p, where beta4-galactosyltransferase (EC 2.4.1.38) had previously been mapped. Consequently, during the evolution of mammals, it is the CKbeta4GT-I gene lineage that has been recruited for the biosynthesis of lactose. CKbeta4GT-II maps to a region of chicken chromosome 8 that exhibits conserved synteny with human chromosome 1p. An inspection of the current human gene map of expressed sequence tags reveals that there is a gene noted to be highly similar to beta4GT located in this syntenic region on human chromosome 1p. Because both the CKbeta4GT-I and CKbeta4GT-II gene lineages are detectable in mammals, duplication of the ancestral beta4-galactosyltransferase gene occurred over 250 million years ago in an ancestral species common to both mammals and birds.

beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.

In murine somatic cells, transcription of the single gene encoding beta 4-galactosyltransferase results in two transcripts of 4.1 and 3.9 kilobases (kb), as a consequence of the use of two transcriptional start sites that are located on exon one separated by 200 base pairs (bp). In early male germ cell development, spermatogonia use only the 4.1-kb start site to yield a transcript that is identical to its somatic cell counterpart. As these cells enter meiosis, there is a switch from the use of this somatic cell start site to the exclusive use, beginning in pachytene spermatocytes, of a male germ cell-specific start site. Germ cell-specific transcripts are distinguished from their somatic counterparts by an additional approximately 560 nucleotides of 5'-untranslated sequence that is located immediately upstream and contiguous with the transcriptional start site defined for the 4.1-kb mRNA (Harduin-Lepers, A., Shaper, N.L., Mahoney, J.A., and Shaper, J.H. (1992) Glycobiology 2, 361-368). This observation predicts the use of a different upstream male germ cell-specific promoter. In this study we show that a 796-bp fragment containing 543 bp of genomic sequence upstream of the germ cell specific transcriptional start site and 253 bp of flanking downstream sequence, directs expression of the reporter gene, beta-galactosidase, exclusively to the pachytene spermatocytes and round spermatids of transgenic mice. This pattern of cell type-specific expression of the transgene is comparable with that of the endogenous beta 4-galactosyltransferase gene.

The beta 1,4-galactosyltransferase (beta 1,4-GT) gene is unusual in that it specifies two mRNAs in somatic cells of 3.9 and 4.1 kilobases (kb). These two transcripts arise as a consequence of initiation at two different sets of start sites that are separated by approximately 200 base pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of their NH2-terminal cytoplasmic domain (Russo, R.N., Shaper, N. L., and Shaper, J.H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we show that the cellular requirements for beta 1,4-GT correlate with the transcriptional start site used. In cells and tissues that express low transcript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site plus low levels of transcription from the 3.9-kb start site result in the intermediate beta 1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactating mammary glands very high transcript levels are observed, which correlate with the predominant use of the 3.9-kb transcriptional start site. To identify the cis-acting elements that regulate the use of the two transcriptional start sites, we constructed a series of beta 1,4-GT/CAT hybrids and carried out transient transfection assays using mouse L cells and Drosophila SL2 cells. These studies have delineated both a distal and proximal regulatory region just upstream of the 4.1- and 3.9-kb transcriptional start sites, respectively. In addition, a negative cis-acting regulatory region was identified that represses transcription from the proximal site. These results suggest a model of transcriptional regulation in which the distal region functions as a housekeeping promoter while the proximal region functions as a mammary cell-specific promoter. Differential initiation from the two promoters is a mechanism for regulation of beta 1,4-GT enzyme levels. The predictions from this model are consistent with the conclusion that both protein isoforms are functionally equivalent resident trans-Golgi membrane-bound enzymes.

The ordered assembly of the carbohydrate moieties of glycoconjugates requires the coordinated actions of a functional family of biosynthetic enzymes, the glycosyltransferases, which are resident membrane-bound proteins of the endoplasmic reticulum and Golgi apparatus. Recent results are beginning to provide insights into the primary structures, the topological organization of specific glycosyltransferases in the endoplasmic reticulum and Golgi, and the protein domain(s) responsible for subcompartmentalization of these membrane-bound enzymes within the Golgi complex.

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.

Beta 1,4-galactosyltransferase (beta 1,4-GT) is a Golgi-resident, type II membrane-bound glycoprotein that functions in the coordinate biosynthesis of complex oligosaccharides. Additionally, beta 1,4-GT has been localized to the cell surface of a variety of cell types and tissues where it is proposed to function in intercellular recognition and/or adhesion. Thus beta 1,4-GT is an appropriate molecule to be used in analyzing the molecular basis for retention of a membrane-bound enzyme in the Golgi complex and its subsequent or alternative transport to the cell surface. Previously we have shown that the gene for bovine and murine beta 1,4-GT is unusual in that it specifies a short (SGT) and long (LGT) form of the enzyme (Russo, R. N., Shaper, N. L., and Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331). The only difference between the two related forms is in the primary structure of the cytoplasmic domains, where LGT has an NH2-terminal extension of 13 amino acids. In this study, we have tested the hypothesis that LGT and SGT are differentially retained in the Golgi or directed to the cell surface. LGT, SGT or chimeric proteins, containing the NH2-terminal cytoplasmic and transmembrane domain of SGT and LGT fused to the cytoplasmic protein pyruvate kinase, were each stably expressed in Chinese hamster ovary cells. Proteins expressed from each construct were localized by immunofluorescence staining exclusively to a perinuclear region, identified as the Golgi by co-localization with wheat germ agglutinin. Furthermore, the subcellular distribution of both SGT and LGT was restricted to the trans-Golgi compartment as assessed by EM immunoelectron microscopy. These data suggest that both forms of beta 1,4-GT are resident trans-Golgi proteins and that an NH2-terminal segment containing the cytoplasmic and transmembrane domains of SGT (39 amino acids) or LGT (52 amino acids) is sufficient for Golgi retention.

We have reported the characterization of a cDNA (clone 31A) encoding bovine alpha 1,3-galactosyltransferase (alpha 1,3-GT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). With the goal of isolating a full-length cDNA encoding murine alpha 1,3-GT we screened a cDNA library with clone 31A and isolated a 3.4-kilobase (kb) alpha 1,3-GT clone (4A). The murine coding sequence is 78% similar to that of the bovine alpha 1,3-GT cDNA, but the "stem" region (defined as the region that links the single transmembrane domain to the catalytic domain) of the murine alpha 1,3-GT encoded by clone 4A, is 31 amino acids shorter than the corresponding region of the bovine alpha 1,3-GT. To screen for heterogeneity in the murine alpha 1,3-GT transcripts, we carried out a polymerase chain reaction (PCR) analysis on mouse C127 cDNA. Four distinct transcripts were detected, which predict four isoforms of the alpha 1,3-GT polypeptide that differ only in the length of their stem region. To determine how the four different transcripts are generated from a single gene, we have established the genomic organization for murine alpha 1,3-GT. The full-length mRNA spans at least 35 kb of genomic DNA and is distributed over nine exons that range in size from 36 base pairs (bp) to approximately 2600 bp. The protein coding region is distributed over six exons, and the 5'-untranslated sequence is distributed over three exons. Comparison of the genomic DNA sequence with that of the four different mRNAs indicates that these transcripts are produced by alternative splicing of the murine pre-mRNA according to a cassette model. A tissue survey using RNA-PCR revealed the presence of four different alpha 1,3-GT transcripts in all mouse tissues and cell lines examined to date, with the notable exception of male germ cells. Additionally, although alpha 1,3-GT levels increased upon thioglycollate-induced activation of mouse peritoneal macrophages, the ratio of the alpha 1,3-GT isoforms was essentially unchanged. Similar results were obtained upon retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. Lastly, a similar PCR analysis of bovine cDNA produced only a single DNA fragment, corresponding to bovine cDNA clone 31A.

UDP-Gal:Gal beta 1----4GlcNAc alpha 1----3-galactosyltransferase is a terminal glycosyltransferase that is widely expressed in a variety of mammalian species, with the notable exception of man, apes, and Old World monkeys. We recently reported the isolation of a bovine cDNA clone that contains the complete coding sequence for this enzyme (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Using this cDNA as a probe, we have demonstrated that, although transcripts cannot be detected in a variety of established human cell lines by Northern blot analysis, homologous sequences are present in human genomic DNA. To establish that these sequences represent a human homologue of alpha 1----3-galactosyltransferase, we have used the bovine cDNA as a probe to isolate two nonoverlapping clones (HGT-2 and HGT-10) from a human genomic DNA library. Clone HGT-2 contains a 1.5-kilobase uninterrupted linear sequence similar to bovine alpha 1----3-galactosyltransferase that is organized as a processed pseudogene. This sequence, flanked by Alu type repeats, contains a short 5'- and 3'-untranslated region and a complete recognizable coding region that is 81% similar at the nucleotide level to bovine alpha 1----3-galactosyltransferase. This putative coding region contains multiple frameshift mutations and nonsense codons in all three reading frames which precludes the synthesis of a functional enzyme. Nevertheless, after optimal alignment, translation predicts a polypeptide that is 68% similar at the amino acid level to the bovine enzyme. Based on Southern analysis and limited sequence analysis, clone HGT-10 contains coding sequences similar to the NH2-terminal region of bovine alpha 1----3-galactosyltransferase. By analysis of panels of human-rodent somatic cell hybrids we have established that the nonfunctional, processed pseudogene and the human homologue represented by HGT-10 are located on human chromosomes 12 and 9, respectively. Interestingly, a comparison of the predicted amino acid sequence of the carboxyl-terminal two-thirds of human alpha 1----3-galactosyltransferase, with the corresponding region of the human blood group A, UDP-GalNAc:[Fuc alpha 1----2]Gal beta 1----4GlcNAc alpha 1----3-GalNAc-transferase (Yamamoto, F., Marken, J., Tsuji, T., White, T., Clausen, H., and Hakomori, S. (1990a) J. Biol. Chem. 265, 1146-1151), reveals a significant similarity (39%) suggesting that these two enzymes may have arisen from the same ancestral gene as a result of gene duplication and subsequent divergence.

The gene for alpha 1----3-galactosyltransferase, termed Ggta-1, was mapped to mouse chromosome 2 by Southern blot analysis of Chinese hamster x mouse somatic cell hybrids. Using an intersubspecies back-cross, this locus was positioned to the centromeric region on this chromosome, near the Hc locus.

Although interleukin 2 (IL 2) clearly up-regulates the expression of the p55 chain of the IL 2 receptor (IL 2R) little is known about its role in the induction of the high-affinity IL 2R. Resting T lymphocytes were induced to express IL 2R under experimental conditions in which IL 2 production was not induced or was prevented. Under these conditions high- and low-affinity IL 2R were easily demonstrated by Scatchard analysis. Northern blot analysis confirmed the accumulation of p55 specific mRNA and the absence of the IL 2 transcript. High-affinity IL 2R induced in the complete absence of IL 2 were unable to transmit a proliferative response unless exposed to extremely high concentrations of IL 2. The addition of picomolar amounts of recombinant IL 2 or the initiation of endogenous IL 2 production during the induction period restored the functionality of high-affinity IL 2R. Also, T cells induced to generate IL 2 displayed functional high-affinity IL 2R even in the presence of monoclonal antibodies blocking extracellular IL 2 and IL 2R. These results indicate that the presence of IL 2 during the early phase of T cell activation is an absolute requirement for the induction of fully operational high-affinity IL 2R and that low amounts of intracellular IL 2 are sufficient to confer functional properties to these receptors. The data also suggest that an intracellular as well as an extracellular high-affinity structure, expressed as a consequence of cell activation, is responsible for conferring competence to the high-affinity IL 2R involved in IL 2-dependent proliferation.

We have used S1 and primer extension analysis to demonstrate that the gene for bovine beta 1----4-galactosyltransferase specifies two sets of mRNA transcripts of different lengths. The longer mRNA transcripts initiate upstream of two in-frame ATG codons and encode a protein of 402 amino acids (long form). The shorter mRNA transcripts initiate between the two in-frame ATG codons and encode a protein of 389 amino acids (short form). These two related forms of beta 1----4-galactosyltransferase have an identical large (358 amino acids), potentially glycosylated, COOH-terminal catalytic domain, and an identical single transmembrane domain. The only difference in primary structure between the two forms is that the long form contains an NH2-terminal extension of 13 amino acids. Thus, bovine beta 1----4-galactosyltransferase fits the pattern established for murine beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. L., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428). Inspection of the NH2-terminal domain suggests that the long form of the bovine enzyme, like its murine counterpart, has a functional cleavable signal sequence which would dictate that the two forms of the membrane-bound enzyme are oriented in opposite directions. We have tested this hypothesis by in vitro translation in the absence or presence of dog pancreas microsomes. In vitro translation of RNA transcripts for the long and short form of beta 1----4-galactosyltransferase in the absence of microsomes results in the synthesis of polypeptides with apparent Mr of 44,500 and 43,000, respectively. In vitro translation of each transcript in the presence of microsomes results in the synthesis of two glycosylated, endoglycosidase H-sensitive proteins with apparent Mr of 47,500 and 46,000. These experiments and additional protease protection experiments demonstrate that the COOH-terminal domain of both the short and the long form of bovine beta 1----4-galactosyltransferase are translocated into the microsomal lumen. By extrapolation, both forms of the enzyme are oriented in vivo as Type II membrane-bound glycoproteins.

Previously we have shown that the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT; UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) is unusual in that it specifies two sets of mRNAs of about 3.9 and 4.1 kilobases (kb). Translation of the 3.9- and 4.1-kb mRNAs results in the predicted synthesis of two related membrane-bound forms of the protein of 386 amino acids (short form) and 399 amino acids (long form), respectively. In this study we have examined the expression of beta 1,4-GT during murine spermatogenesis. Spermatogonia contain a 4.1-kb transcript that is comparable in size to the beta 1,4-GT mRNA identified in somatic cells. During differentiation from spermatogonia (2n) to pachytene spermatocytes (4n), the amount of beta 1,4-GT mRNA is reduced to barely detectable levels. Continued differentiation to round spermatids (n) is coincident with a renewed production of beta 1,4-GT mRNA to levels comparable with those detected in spermatogonia. However, the characteristic 4.1-kb mRNA detected in spermatogonia is replaced by two truncated transcripts of 2.9 and 3.1 kb. By S1 nuclease analysis, the 2.9- and 3.1-kb transcripts were shown to encode the same open reading frame as the 4.1-kb transcript found in somatic cells. The shorter round spermatid transcripts arise as a consequence of the use of alternative poly(A) signals. Lastly, we show that, in direct contrast to all somatic tissues and cell lines examined to date, male germ cells synthesize only the long form of the beta 1,4-GT polypeptide.

To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. Ricin(II)-agarose lectin affinity chromatography, oligomapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the beta4GalT-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an approximately 2-4-fold reduction in the level of transcripts of all six beta4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian beta4GalT genes.

The enzyme β4-galactosyltransferase-I (β4GalT-I; UDP-Gal:GlcNAc β4-galactosyltransferase; EC 2.4.1.38) is a constitutively expressed, trans-Golgi resident, type II membrane-bound glycoprotein that is widely distributed in vertebrates. The protein domain structure established for β4GalT-I consists of: (1) a short NH2-terminal cytoplasmic domain of 11 or 24 amino acids depending on the protein isoform (Shaper et al. 1988; Russo et al. 1990); (2) a large COOH-terminal luminal domain containing the catalytic center (~270 amino acids) linked to a single transmembrane domain (19 amino acids) through a glycosylated peptide segment (~86 amino acids) termed the stem region. In essentially all vertebrate tissues, the primary function of β4GalT-I is to catalyze the transfer of Gal from UDP-Gal to GlcNAcβ-R, forming the N-acetyllactosamine (Galβ1-4GlcNAcβ1-R) or poly-N-acetyllactosamine structures assembled on glycoconjugates.

Cell surface glycoconjugates (glycoproteins and glycolipids) are well positioned topologically to be involved in cell-cell interactions and transmembrane signaling. Their glycans (sugar chains) protrude from the surface, and display considerable structural complexity. The glycan structures assembled in a given cell type are dictated in part by the subset of glycosyltransferases that are translationally expressed. As the relative levels of the various glycosyltransferases determine which carbohydrate structures are carried by eukaryotic glycoconjugates, they indirectly affect intercellular interactions. In particular, the terminal sugars of N-and O-linked glycans are known to play a role in receptor-mediated recognition and binding.

β1,4-Galactosyltransferase (β4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian β4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by ≈200 bp. In the mammary gland, coincident with the increased β4GalT-I enzyme level (≈50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a β4GalT-I transcript in which the 5′- untranslated region (UTR) has been truncated from ≈175 nt to ≈28 nt. The 5′-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5′-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5′-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo . The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (≈14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, β4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

Glycoconjugates (glycolipids and glycoproteins) are a major class of biomolecules found on the cell surface of essentially all cells and tissues. A remarkable and somewhat bewildering number of different oligosaccharide structures (glycans) have been isolated and characterized from glycoconjugates. Because of this remarkable structural diversity, oligosaccharide structures have long been considered ideal candidates for information-containing molecules which can mediate intercellular communication and recognition or alternatively, communication between a cell and its environment. Indeed, ordered, sequential changes in the expression of specific glycan structures at the cell surface may provide an important mechanism for controlling cell-cell behavior and fate during development. During early murine embryogenesis, temporal changes in cell surface glycosylation patterns (stage specific antigens) have been documented using monoclonal antibodies that are specific for defined glycan structures (reviewed in 1).

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.

alpha-1,3-Galactosyltransferase is a terminal glycosyltransferase that is widely expressed in a variety of mammalian species, with the notable exception of man, apes, and Old World monkeys. Although transcripts for this enzyme are not detectable in humans, homologous sequences have been identified in human genomic DNA. These sequences correspond to a processed pseudogene that maps to chromosome 12 and the inactivated remnant of the once functional source gene that maps to chromosome 9. We have now established that the former sequence (GGTA1P) is localized to 12q14-q15 and the latter sequence (GGTA1) is localized to 9q33-q34 [corrected].

We have previously shown that the expression of the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT, UDP-galactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D galactosyltransferase, EC 2.4.1.38) is fundamentally different between somatic and male germ cells (Shaper et al., 1990b). In somatic cells, two transcripts of 3.9 kb and 4.1 kb are produced. In contrast, in spermatogonia only the 4.1 kb transcript is expressed. Maturation of spermatogonia to pachytene spermatocytes is accompanied by reduced expression of the 4.1 kb transcript to barely detectable levels. Continued differentiation to haploid round spermatids is coincident with renewed expression in which the 4.1 kb transcript is replaced by two truncated transcripts of 2.9 and 3.1 kb. In this study, we report the characterization of a full-length beta 1,4-GT cDNA clone from a murine round spermatid library that corresponds to the 2.9 kb transcript. This transcript encodes the same open reading frame as the 4.1 kb transcript, but utilizes alternative poly(A) signals embedded within the long 3'-untranslated region of the somatic transcript. Based on sequence analysis, together with primer extension and S1 nuclease protection experiments, both the 2.9 and the 3.1 kb round spermatid beta 1,4-GT transcripts are distinguished by the presence of an additional 5'-untranslated sequence of approximately 560 bp that is absent in premeiotic germ cells and somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Gal beta 1----4GlcNAc alpha 1----3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J.Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active alpha 1----3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant alpha 1----3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine-Sepharose. The recombinant alpha 1----3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Gal beta 1----4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus alpha 1----3-galactosyltransferase [Blanken, W. M.Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927-12934]. In pilot studies, the recombinant enzyme was examined for the ability to synthesize alpha 1----3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H-NMR, methylation analysis, HPLC, and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Gal alpha 1----3Gal beta 1----4-R. Our results demonstrate that catalysis by recombinant alpha 1----3-galactosyltransferase can be used to obtain preparative quantities of various alpha 1----3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

A 1.3-kilobase cDNA clone (7A) coding for bovine galactosyltransferase (glycoprotein 4-beta-galactosyltransferase, EC 2.4.1.38) was isolated from a lambda gt11 expression library by immunological screening with monospecific polyclonal antisera to the affinity-purified bovine enzyme. The nucleotide sequence of this clone predicts an open reading frame that starts at the 5' end of the insert and codes for a polypeptide of 334 amino acids with Mr 37,645. Based on a Mr of 57,000 for the membrane-bound enzyme this clone accounts for approximately 61% of the coding sequence. Portions of the predicted amino acid sequence matched the six tryptic peptides isolated from affinity-purified bovine galactosyltransferase. Clone 7A hybridizes to a 4.8-kilobase bovine mRNA and identifies multiple EcoRI restriction fragments in bovine, murine, and human DNA.

We have isolated overlapping cDNA clones representing the full length (4038 base pairs) transcript for murine beta-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with three distinct structural features, a large COOH-terminal domain (355 amino acids), a single transmembrane domain (20 amino acids), and a short NH2-terminal domain. Primer extension analysis, S1 protection analysis, and RNA blotting demonstrate the presence of two sets of mRNA transcripts which differ in length by about 200 base pairs. The 5' boundary of the long transcripts maps upstream of two in-frame ATGs. The 5' boundary of the short transcripts maps between these two ATGs. These results predict that two related forms of beta-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. The long form (399 amino acids) has an NH2-terminal extension of 13 amino acids.

Pyrimidine dimer formation in response to UV radiation is governed by the thymine content of the potential dimer and the two flanking nucleotides. An enzymatic activity can be purified from Micrococcus luteus that cleaves the N-glycosyl bond between the 5' pyrimidine of a dimer and the corresponding sugar without rupture of a phosphodiester bond. We propose that strand scission at a dimer site by the M. luteus enzyme requires two activities, a pyrimidine dimer DNA-glycosylase and an apyrimidinic/apurinic endonuclease.

The mechanism of action of the homogeneous preparation of human placental apurinic/apyrimidinic (AP) endonuclease, described in the previous paper (Shaper, N. L., Grafstrom, R. H., and Grossman, L. (1982) J. Biol. Chem. 257, 13455-13458), has been investigated in detail. This enzyme cleaves apyrimidinic DNA both 5' and 3' to the site of damage in a ratio of 60:40, respectively. Even though this enzyme can cleave on both sides of an internal AP site, it does not release deoxyribose 5-phosphate from terminal AP sites. However, a compound, tentatively identified as alpha, beta unsaturated deoxyribose 5-phosphate, is nonenzymatically released only from 5'-terminal AP sites, presumably by a beta-elimination mechanism.

An endonuclease from human placenta has been purified to apparent homogeneity, which acts specifically on DNA containing either apurinic or apyrimidinic sites. The isolation procedure, which results in a 20,000-fold purification and an overall yield of 15%, employs chromatography on a gel of octyl succinic anhydride coupled to agarose by diaminohexane spacers, isoelectric focusing, Sephadex G-75 chromatography, and DNA agarose affinity chromatography. Under conditions in which proteolysis is minimized, this enzyme appears to be the major species of apurinic/apyrimidinic endonuclease. The endonuclease is a monomeric protein with an apparent Mr = 37,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 7.4-7.6, requires Mg2+, is partially stimulated by Mn2+, and is inhibited by EDTA. It has no detectable exonuclease or phosphomonoesterase activity.

We have isolated, by immunological screening of a lambda gt11 expression library, a cDNA clone that represents the complete coding sequence for bovine alpha 1----3-galactosyltransferase. The coding sequence predicts a membrane-bound protein with three distinct structural features: a large, potentially glycosylated COOH-terminal domain (346 amino acids), a single transmembrane domain (16 amino acids), and a short NH2-terminal domain (6 amino acids). Thus, the domain structure for this transferase is similar to that deduced for beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428) and alpha 2----6-sialyltransferase (Weinstein, J., Lee, E. V., McEntee, K., Lai, P.-H., and Paulson, J. C. (1987) J. Biol. Chem. 262, 17735-17743). S1 analysis demonstrates that two sets of mRNAs, which are heterogeneous at their 5' ends, are transcribed. Because both sets initiate upstream of the translational start site, only one protein is encoded by this gene. alpha 1----3-Galactosyltransferase is widely expressed in different mammalian species, with the notable exception of man and Old World monkeys (Galili, U., Shohet, S. B., Kobrin, E., Stults, C.L.M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762). By Northern blot analysis we were indeed unable to detect transcripts for this enzyme in various human and Old World monkey cell lines; transcripts were readily detected in other mammalian species. However, by Southern blot analysis, homologous sequences for alpha 1----3-galactosyltransferase were identified in human genomic DNA. This suggests that the gene, although present in the human genome, is normally not expressed. These observations have potential medical implications. Because many humans have high levels of circulating antibodies directed against the enzymatic product of alpha 1----3-galactosyltransferase (Gal alpha 1----3Gal beta 1----4GlcN Ac) (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373), it has been suggested that activation of this normally silent gene may play a role in autoimmune disease in man (Etienne-Decerf, J., Malaise, M., Mahieu, P., and Winand, R. (1987) Acta Endocrinol. 115, 67-74).

An essential step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked glycans on zona pellucida glycoprotein 3 (ZP3). While there is agreement on the primary role of O-linked glycans in sperm-ZP3 binding, there is a striking lack of consensus on both the terminal monosaccharide(s) required for a functional binding site and the cognate protein on the sperm cell surface that recognizes this glycan. Much current debate centers on the essential role of nonreducing terminal N-acetyl-glucosaminyl or alternatively, alpha-galactosyl residues, to form a functional sperm binding ligand. Relevant to this debate, we demonstrated that alpha 1,3-galactosyltransferase (alpha 3-GT), which adds nonreducing terminal alpha-galactosyl residues to glycans, is not expressed in murine spermatocytes or spermatids. The objectives of this study were to determine whether alpha 3-GT is expressed in female germ cells and to compare the pattern of expression of two other terminal glycosyltransferases, beta 1,4-galactosyltransferase (beta 4-GT) and alpha 2,6-sialyltransferase (alpha 6-ST), between male and female germ cells. Total RNA was isolated from growing oocytes obtained from 15-day-old animals, fully grown oocytes, and eggs as well as spermatogonia, spermatocytes, and spermatids. The presence of alpha 3-GT, beta 4-GT, and alpha 6-ST mRNAs was analyzed by an RT-PCR-based assay. Our data demonstrate that the alpha 3-GT gene is expressed in female germ cells, but not in male germ cells. In contrast, both beta 4-GT and alpha 6-ST are expressed during oogenesis and spermatogenesis. This differential expression of alpha 3-GT in female germ cells is consistent with the model of sperm-egg binding in which a nonreducing terminal alpha-galactosyl residue is required for a functional determinant on ZP3 and with our hypothesis that the biological significance for the suppression of alpha 3-GT expression in male germ cells is to prevent sperm-sperm aggregation.

A cell surface UDP-galactose:N-acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity-purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti-GT antibody resulted in its time-dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.

The structural gene for galactosyltransferase (glycoprotein 4-B-galactosyltransferase, EC 2.4.1.38) was localized to human chromosome 9 band p13 by chromosome in situ hybridization using a cloned bovine galactosyltransferase cDNA probe. This chromosomal location is at the same position to which galactose-1-phosphate uridyltransferase, an enzyme which provides the nucleotide sugar substrate (UDP-galactose) for galactosyltransferase, has been mapped.

We have isolated overlapping cDNA clones representing the full-length transcript (4038 base pairs) for murine β-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with 3 distinct structural features: 1) a large, potentially glycosylated COOH-terminal domain (355 amino acids) which is positioned within the Golgi lumen and contains both the catalytic and α-lactalbumin binding site; 2) a single transmembrane domain (20 amino acids); and 3) a short NH 2 -terminal domain containing 2 Met residues, separated by 12 amino acids. The gene for murine β-1,4-galactosyltransferase is unusual in that it specifies 2 mRNA transcripts which differ in length by about 200 base pairs. The longer transcript contains both Met residues found in the NH 2 -terminal domain; the shorter transcript contains only the downstream Met. These results predict that 2 related forms of β-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. Both forms of the enzyme are identical in primary structure with the exception that the long form has an NH 2 -terminal extension of 13 amino acids which, in part, potentially encodes a cleavable signal sequence. The structural implications, topological distribution and potential biological significance of the 2 forms of the enzyme are discussed.

Publisher Summary This chapter discusses the purification procedure and properties of the human placental apurinic/apyrimidinic endonuclease. Apurinic/apyrimidinic endonuclease (AP endonuclease) participates in an excision repair mechanism by catalyzing the hydrolysis of phosphodiester bonds in DNA at apurinic or apyrimidinic sites. This enzyme isolated from prokaryotic cells has been purified to apparent homogeneity and chemically characterized; however, it has only been partially purified from eukaryotic cells. The chapter describes the purification of AP endonuclease to apparent homogeneity from human placenta. Assay methods are described. The assay mixture is incubated at 37 ° for 10 min. Although depurinated DNA is itself susceptible to alkaline hydrolysis at the apurinic site, this is a slow reaction which requires 4-hr incubation at room temperature to reach completion. In addition, the average number of apurinic sites per DNA molecule was determined by incubating the apurinic DNA in the denaturation buffer for 4 hr at room temperature. After renaturation and filtration, the number of nicks introduced was determined by Poisson analysis.

In Micrococcus luteus the incision event during repair of DNA containing pyrimidine dimers has been shown in vitro to be a two-step enzymatic reaction. The first step results in the cleavage of the N-glycosylic bond between the 5′-thymine moiety of the pyrimidine dimer and the deoxyribose generating an apyrimidinic site (Apy) and a thymine-thymidylate dimer attached to the DNA. The second catalytic step involves phosphodiester bond hydrolysis 3′ to the Apy site generating a nicked DNA with an Apy site at its 3′ terminus and the mixed thymine-thymidylate dimer at the 5′ terminus.

We have isolated a series of overlapping murine genomic DNA clones that include the complete coding sequence of the Golgi membrane bound marker enzyme β-1,4-galactosyltransferase. The coding sequence is distributed into six exons spanning 50,000 b.p. of mouse chromosome 4. The COOH terminal domain is predominantly encoded by exons 2–6 and the transmembrane and amino terminal cytoplasmic domains are encoded by exon 1. S1 analysis establishes the most 5′ transcriptional initiation site 190 b.p. upstream of the first methionine residue.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.