Targeted mutations in β1,4-galactosyltransferase I reveal its multiple cellular functions

Biochimica et Biophysica Acta 1573 (2002) 258 – 270 www.bba-direct.com

Review

Targeted mutations in h1,4-galactosyltransferase I reveal its multiple cellular functions Carey Rodeheffer, Barry D. Shur * Department of Cell Biology, Emory University School of Medicine, Room # 405, 615 Michael Street, Atlanta, GA 30322, USA Received 31 October 2001; accepted 1 May 2002

Abstract h1,4-Galactosyltransferase I (GalT I) is one of the most extensively studied glycosyltransferases. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosaccharide chains on glycoproteins and glycolipids. GalT I has also been reported in non-Golgi locations, most notably the cell surface, where it has been suggested to function nonbiosynthetically as a receptor for extracellular glycoside substrates. Cloning of the GalT I cDNAs revealed that the gene encodes two similar proteins that differ only in the length of their cytoplasmic domains. Whether these different GalT I proteins, or isoforms, have similar or different biological roles is a matter of active investigation. The functions of the GalT I proteins have been addressed by targeted mutations that eliminate either both GalT I isoforms or just the long GalT I isoform. Eliminating both GalT I proteins abolishes most, but not all, GalT activity, an observation that led to the realization that other GalT family members must exist. The loss of both GalT I isoforms leads to neonatal lethality due to a wide range of phenotypic abnormalities that are most likely the result of decreased galactosylation. When the long isoform of GalT I is eliminated, galactosylation proceeds grossly normal via the short GalT I isoform, but specific defects in cell interactions occur that are thought to depend upon a non-biosynthetic function of the long GalT I isoform. D 2002 Elsevier Science B.V. All rights reserved. Keywords: h1,4-Galactosyltransferase I; Glycosylation; Golgi complex; Plasma membrane; Fertilization; Mammary gland

1. Introduction h1,4-galactosyltransferase I (GalT I) is one of the most exhaustively studied of the glycosyltransferases. It was one of the first, if not the first, glycosyltransferases to be purified to apparent homogeneity [1], the first mammalian glycosyltransferase cDNA to be cloned [2 – 4], and the first mammalian glycosyltransferase to have its crystal structure resolved [5,6]. GalT I is most often thought of as a constituent of the trans-Golgi apparatus, where it transfers galactose (Gal) in a h1,4-linkage from UDP-Gal to terminal N-acetylglucosamine (GlcNAc) residues on elongating oligosaccharide chains. The abundance of GalT I in the Golgi complex has made it a convenient marker for this organelle resulting from either subcellular fractionation or indirect immunofluorescence [7 –9]. GalT I is also present in soluble form in

*

Corresponding author. Tel.: +1-404-727-4315; fax: +1-404-727-6256. E-mail address: [email protected] (B.D. Shur).

many bodily fluids, including serum, cerebrospinal fluid, vitreous humor, amniotic fluid, colostrum, and milk, from which it was initially purified as part of the lactose synthetase complex [1,10]. In addition to its traditional localization in the Golgi complex and its presence in a variety of body fluids, GalT I has also been detected on the plasma membrane of specific cell types [11]. As is the case for the soluble forms of GalT I, the function of the cell surface GalT I is not entirely clear. There are data to indicate that GalT I, as well as other glycosyltransferases, may function on the cell surface as receptors for extracellular glycoside ligands. Since sugar nucleotide donors are not normally present in the extracellular fluids, it has been suggested that the surface-localized glycosyltransferase would form a stable adhesive bond with its glycoside substrate in the extracellular matrix or on an adjacent cell surface [11 –14]. The initial reports of glycosyltransferases on the cell surface were controversial since it was difficult to completely eliminate all possible contamination from intracellular sources and the early immunohistochemistry stud-

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ies relied upon antibodies with questionable specificity [7,10,15,16]. Nevertheless, reports from many workers using a battery of model systems and well-defined antibodies have confirmed the presence of some glycosyltransferases, most notably GalT I, on the plasma membrane of specific cell types [17 – 21]. Recent structural analysis of GalT I demonstrates its ability to bind its glycoside substrate in the absence of sugar nucleotide and adopt a stable, lectin-like conformation [6]. Unlike GalT I expression in the Golgi complex, which is characteristic of virtually all higher eukaryotic cells, the expression of GalT I on the cell surface ranges dramatically. On some cells, such as sperm, all cellular GalT I is expressed as an integral membrane protein on the plasma membrane [18]. In contrast, the amount of GalT I protein on the surface of somatic cells is below the resolution of detection by most immunohistochemistry, as compared with the vast majority of GalT I (>90%) concentrated in the Golgi complex. Consequently, GalT I is only readily detectable on the surface of somatic cells when the assay conditions have been optimized for its expression. Finally, some cell types, if not most, appear to be devoid of any GalT I on their surfaces [17]. Although most work has focused on the presence and function of GalT I on the cell surface, recent reports have documented the presence of other surface glycosyltransferases, including polysialyltransferases [22], fucosyltransferase I (FUCTI) [23], (possibly fucosyltransferase V [24]), an N-acetylgalactosaminyltransferase III [25], and an Nacetylgalactosaminyl-phospho-transferase [26]. Among these, there are strong data to suggest that GalT I, FUCTI, and N-acetylgalactosaminyl-phospho-transferase do indeed function as surface receptors for extracellular glycoside ligands. The near ubiquity of GalT I in the Golgi complex of higher eukaryotes and its potential to function non-biosynthetically in other subcellular domains, has focused intense interest on this protein. Insight into these issues first came from the cloning of the GalT I cDNAs, which eventually enabled the production of mice bearing targeted deletions in the GalT I proteins.

2. Molecular biology of GalT I The gene for GalT I encodes two similar, but not identical, proteins [27 – 29]. Both GalT I proteins have a type II membrane conformation, analogous to all other glycosyltransferases cloned to date [30], with a relatively short amino-terminal cytoplasmic domain, a signal sequence/transmembrane domain, and a large carboxy-terminal lumenal or extracellular catalytic domain (Fig. 1). The two GalT I proteins have identical catalytic and transmembrane domains but differ in their cytoplasmic domains due to translation from two different in-frame AUGs that result from differential transcription initiation of two RNAs [27 –

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29]. The shorter protein has a cytoplasmic tail of only 11 amino acids, whereas the longer species has a 24-aminoacid cytoplasmic domain. 2.1. Do the two different GalT I isoforms have similar or unique functions? Whether the two different GalT I isoforms have functionally distinct roles within the cell or, rather, reflect tissuespecific proteins with similar, if not identical, biosynthetic functions remains an issue of active debate [11]. There are currently two principle schools of thought regarding the functions of the two isoforms, though they are not mutually exclusive. Studies by Shapers et al. have suggested that the two isoforms result from differential transcriptional and translational controls, which have evolved to accommodate the rapid increase in GalT I activity that occurs during lactation. GalT I mRNA levels rise 10-fold during lactation, and within this pool, the transcript encoding the short GalT I isoform is 10 times more abundant than the long GalT I transcript. The specific upregulation of the short isoform is suggested to occur through the concerted action of specific positive and negative transcription factors as well as by a more rapid rate of translation, relative to the long isoform. The 5V-UTR of the transcript encoding the short isoform does not have the secondary structure seen in the 5V-UTR of the long transcript, which is thought to impede translational efficiency [31 –33]. A second body of work, mostly from Shur et al. [11] and Evans et al. [34], has suggested that the long isoform, in addition to its biosynthetic role in the Golgi complex, can also function as a signal transducing receptor on the cell surface. This is thought to result from the additional amino acid residues that distinguish the long and short GalT I cytoplasmic domains from one another. Although it is clear that the long and short isoforms function biosynthetically in the Golgi complex, there are data to indicate that a portion of the long isoform is also expressed on the surface of some cell types where it binds glycoside substrates under noncatalytic conditions. Ligand (i.e., substrate)-induced aggregation of GalT I on the cell surface leads to activation of cell type-specific signal transduction cascades. Mutagenesis of specific residues unique to the long cytoplasmic domain inhibits ligand-induced signal transduction by GalT I [35]. In contrast to the long isoform, it is suggested that the short GalT I isoform has a purely biosynthetic function. Even if ectopically expressed on the cell surface, the short GalT I isoform is devoid of the cytoplasmic domain sequences required for ligand-induced signal transduction [35,36]. Since the transmembrane domain of GalT I is critical for its retention in the Golgi complex [37 – 42], it remains unclear how any GalT I protein is able to override the Golgi retention signal and transit to the cell surface. The long GalT I cytoplasmic domain may somehow modulate the ability of the transmembrane domain to retain the protein in the Golgi

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Fig. 1. The genomic organization for the murine GalT I gene. The gene for GalT I encodes two similar, but not identical proteins. Both GalT I proteins have a type II membrane conformation with a relatively short amino-terminal cytoplasmic domain (C), a signal sequence/transmembrane domain (TM), and a large carboxy-terminal lumenal or extracellular catalytic domain. The two GalT I proteins differ in their cytoplasmic domains due to translation from two different inframe AUGs that result from differential transcription initiation of two RNAs (arrows). The shorter protein has a cytoplasmic tail of only 11 amino acids, whereas the longer species has an additional 13-amino-acid extension in its cytoplasmic domain.

compartment. This must be a regulated, as opposed to a constitutive, event, since only a portion of the long GalT I localizes to the surface (except in sperm), and this occurs in a cell type-specific and ECM-specific manner. As one of many possibilities, phosphorylation of the cytoplasmic domain may influence its binding to effector proteins that in turn influence Golgi retention and/or exit. HeLa cell GalT I has, in fact, been shown to be serine phosphorylated, presumably within the cytoplasmic domain [43], but its physiological significance remains unknown as is the identity of the phosphorylated GalT I isoform(s).

3. Two types of targeted mutations in GalT I have been characterized The ability to create targeted deletions in specific genes has enabled investigators to examine the isoform-specific function of the long and short GalT I proteins. Two types of mice have been created bearing targeted mutations in GalT I [36,44,45]. The first mutation was made by replacing exon 1 with a neo selection cassette. Exon I encodes the cytoplas-

mic domain, transmembrane domain, and a portion of the catalytic domain for the long and short isoforms of GalT I. Therefore, these mice are null for both GalT I isoforms (total GalT I knockout). As discussed below, elimination of both GalT I isoforms leads to a neonatal lethal phenotype, presumably due to a loss of critical h1,4-galactosyl residues [44,45]. Since total GalT I knockout mice are null for both GalT I isoforms, long and short, they do not shed light on the differential function, if any, of the two GalT I isoforms. Thus, phenotypes due to deficiencies in galactosylation cannot be distinguished from phenotypes that might reflect non-biosynthetic functions of GalT I. To address this issue, a second, more specific targeted mutation was created that eliminated only the long GalT I isoform (long GalT I knockout) [36]. Rather than replacing the first exon with a neo selection cassette, exon 1 was replaced with one in which the first ATG was mutated. Thus, these animals can only synthesize the short isoform of GalT I. They would be unable to initiate translation from the mutagenized ATG and should be devoid of the long GalT I isoform. The production of this second line of knockout mice successfully separated,

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for the first time, the function of GalT I during glycoconjugate biosynthesis from its proposed non-biosynthetic function as a signal-transducing receptor.

4. Null mutations in both GalT I isoforms 4.1. Effects on galactosyltransferase enzyme activity Two independent groups simultaneously reported the phenotype of the total GalT I-null animal (Table 1). Both Table 1 Phenotypes of GalT I-null mice Phenotypes of total GalT I-null mice Enzyme activity: . GalT activity is absent in most tissues. . Brain, testis, liver, and other organs have varying levels of residual GalT activity. Galactosylated products: . The Galh-1,4-GlcNAc epitope is reduced in glycoconjugates from testis, heart, intestine, lung, and serum. . Galh1,4-GlcNAc epitope expressed normally in brain. . Ninety percent reduction in terminal h1,4-galactosyl residues on N-linked glycans of serum glycoproteins, and to a lesser extent on N-linked glycans from erythrocyte membranes. . Biantennary complex-type oligosaccharides are completely eliminated. . Eighty percent reduction in galactosylation of core 2 O-linked oligosaccharides from erythrocyte membrane glycoproteins.

Reference

4.2. Effects on the expression of b1,4-galactose residues

[44 – 46]

[48] [Izui et al., unpublished results]

[51]

Reference

Enzyme activity: . GalT activity is near normal in cell types assayed: [36,54] spermatogenic cells and mammary gland. Galactosylated products: . Galh1,4-GlcNAc epitopes are similar in wild-type [44] and mutant testis by RCA lectin blotting. Biological effects: . Sperm are unable to bind the egg coat ligand, or undergo egg-induced acrosome reactions. . Mammary epithelial cells show defective interactions with the basal lamina matrix, leading to abnormal branching morphogenesis and gland differentiation.

groups demonstrated a global reduction, but not elimination, of GalT activity toward GlcNAc-containing substrates in tissue lysates. Little or no activity was detected in the salivary gland, stomach, adrenal gland, intestine, pancreas, spleen, lung, thymus, liver, kidney, heart, or muscle of the null animal [44,45]. However, the GalT I-null testis contained 4% of the activity in wild-type testis [44] and brain contained 30% [44] or 65% of wild-type activity [46]. The products of the residual galactosyltransferase activities were sensitive to both bovine galactosidase (recognizes Galh1,4GlcNAc, Galh1,6-GlcNAc and Galh1,3-GlcNAc linkages) and diplococcal galactosidase (recognizes Galh1,4-GlcNAc linkages) demonstrating that they are indeed products of enzymes with GalT activity [44,46]. The subcellular distribution of the residual GalT activity in testis and brain was not determined.

[44 – 46]

Biological effects: . Animals die in the neonatal period likely due to [44] polyglandular endocrine insufficiency resulting from ungalactosylated anterior pituitary hormones. . Skin and intestinal epithelial cells exhibit [45] augmented proliferation and abnormal differentiation. . Sperm are unable to bind the egg coat ligand, [36] or undergo egg-induced acrosome reactions. Phenotypes of long GalT I-null mice

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[36] [54]

4.2.1. Tissue glycoconjugates Two reagents were used to assess the expression of the GalT I disaccharide reaction product, Galh1,4-GlcNAc: the lectin RCA-I and a2,6-sialyltransferase, which recognizes h1,4-galactosyl groups as its acceptor substrate. As expected, most tissues showed a loss of terminal h1,4galactosyl residues, including testis, heart, intestine, lung [44], as well as serum glycoproteins [45,46]. The possibility that h1,4-galactosyl residues were masked by sialic acid moieties was tested by pretreatment with neuraminidase and subsequent RCA-I lectin blotting [44]. Neuraminidase treatment had no effect on RCA-I reactivity in testis, heart and intestine. Similar results were found when a2,6-sialyltransferase was used to assess the occurrence of h1,4-galactosyl residues [44]. Although most tissues have a dramatic loss of terminal h1,4-galactose, some GalT I-null tissues continue to express h1,4-galactosyl residues, such as brain and salivary gland, which have wild-type levels of reactivity toward RCA-I and a2,6-sialyltransferase, respectively, and lung, which is reactive to RCA-I following neuraminidase treatment [44,46]. Taken together, these data suggest that alternate pathways for h1,4-galactosylation exist in lung, salivary gland and brain, if not in other tissues as well. In subsequent studies, transcripts of other GalT family members have been detected in brain and may be responsible for the GalT Iindependent galactosylation that occurs in this tissue [47]. 4.2.2. Serum glycoproteins The terminal h1,4-galactose moiety on serum glycoproteins has been implicated in the clearance of serum glycoproteins from the circulation by binding to the hepatic asialoglycoprotein receptor (ASGPR) [48,83]. It was predicted, therefore, that the protein concentration in serum from GalT I-null animals would be greater than that in wild-type serum, due to the increased half-life of circulating glycoproteins devoid of terminal h1,4-galactosyl residues. In fact, the

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serum protein concentrations are identical in wild-type and GalT I-null animals, although the serum glycoproteins from the GalT I-null lack terminal galactosyl residues as detected by RCA-I reactivity [48]. N-linked glycans were released from the serum glycoproteins and fractionated by lectin affinity chromatography. The serum glycoproteins from wild-type and GalT I-null animals contain equivalent quantities of N-linked glycans, but only 7.7% of them contain h1,4galactosyl groups in the GalT I-null, as compared with 77% galactosylation of N-glycans in wild type. The defect in terminal galactosylation is most pronounced within the biantennary complex-type oligosaccharides, which are completely devoid of h1,4-galactosyl residues [48]. These results suggest that the clearance of serum glycoproteins is dependent upon mechanisms other than recognition of terminal h1,4-galactosyl residues by the ASGPR. Consistent with this, targeted deletion of the ASGPR has no significant effect on the levels of circulating serum glycoproteins [83], similar to that seen in GalT I-null animals. It is well known that altered galactosylation of IgG is associated with altered antibody function, and patients with rheumatoid arthritis have reduced galactosylation of IgG and reduced GalT activity [49]. Similar to that reported above for serum glycoproteins in general, IgG from GalT Inull mice is still galactosylated, but the proportion of digalactosylated glycoforms is significantly decreased [Izui et al., unpublished results]. Interestingly, the pattern of IgG galactosylation in GalT I-null mice is very similar to that seen in MRL-lpr/lpr mice, a murine model for lupus, and also mirrors the defects seen on the pathogenic anti-IgG2a rheumatoid factor [50]. The significance of the defective pattern of galactosylation in GalT I-null mice and its relationship to antibody and immune function, if any, is under investigation. 4.2.3. Erythrocyte membrane glycoproteins Similar to that seen in tissue lysates and serum glycoproteins, membrane glycoproteins isolated from GalT Inull erythrocytes are also characterized by reduced galactosylation. Erythrocyte membrane oligosaccharides were fractionated by anion-exchange chromatography followed by pulsed amperometric and fluorescence detection. Galactosylation of core 2 O-linked oligosaccharides is reduced by 80%; galactosylation of N-glycans is reduced by 40%. The decrease in galactosylation leads to a marked reduc-

tion in the presence of sialylated Galh1,4-GlcNAc structures [51]. These results, along with those discussed above, indicate that although galactosylation is reduced in the GalT I-null animal, it is not completely eliminated, further arguing for the existence of alternate pathways of galactosylation. 4.3. The neonatal lethal phenotype is associated with polyglandular endocrine insufficiency The GalT I-null animal exhibits a neonatal lethal phenotype, presumably as a consequence of reduced h1,4-galactosylation of critical glycoprotein (and glycolipid?) structures required for postnatal development. Although both groups of investigators who prepared GalT I-null mice reported a neonatal lethal phenotype, their interpretations of the underlying defects differ. Lu et al. [44] reported that 90% of the GalT I-null animals die within 2 –3 weeks following birth and are characterized by stunted growth, thin skin, sparse hair, and dehydration. At the histological level, the density of hair follicles and subepidermal adipose tissue is reduced, spermatogenesis is delayed, the lung parenchyma is poorly differentiated, and the adrenal cortex is poorly stratified (Fig. 2). The few surviving adults (10%) have puffy skin, difficulty delivering pups at birth, and fail to lactate. All of these defects are consistent with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The polyglandular nature of the endocrine insufficiency is indicative of dysfunction of the anterior pituitary gland. Previous in vitro studies have suggested that proper glycosylation of the anterior pituitary hormones is required for their normal folding, secretion, ability to activate receptors, and clearance [52,53]. The glycoprotein hormones contain both traditional sialylated N-linked oligosaccharides (NeuAca2,3(6)Galh1,4GlcNAch) as well as a unique sulfated oligosaccharide motif (SO4-4GalNAch1,4GlcNAch); the relative distribution of these two N-linked oligosaccharides show both hormone- and species-specificity. Both oligosaccharides have been implicated in hormone activity [52,53]; for example, the sialylated chains may be involved in modulating hormone activation of target receptors, whereas the sulfated glycan may influence hormone half-life via recognition by the hepatic Man/GalNAc-4-SO4 receptor. In any event, it is not surprising that altering the carbohydrate

Fig. 2. Phenotypic analysis of GalT I-deficient mice. (a,b) Views of 3-week-old wild-type and GalT I / C57BL/6J black and albino mice. The GalT I / mice (right of each pair) are dwarf with sparse hair and thin skin. Note the intestinal contents visible through the albino skin as is the scalp; the hair is missing on the dorsal neck of the black GalT I / animal. (c,d) Sections through age-matched dorsal, thoracic-lumbar skin from wild-type (c) and GalT I / (d) mice. Note the reduced development of hair follicles and subdermal adipose tissue. (e,f) Sections of testis from wild-type (e) and GalT I / (f) males. Spermatogenesis is delayed in the mutant as evidenced by the smaller seminiferous tubules and by the predominance of pachytene spermatocytes in GalT I / mice (arrowhead) relative to the predominance of round and late spermatids in wild type (arrowhead). (g,h) Sections through lungs of newborn wild-type (g) and GalT I / (h) mice. Note the thick trabeculae in GalT I / and the smaller alveoli as compared to wild type. (i) Views of dissected whole adrenal glands from wild-type (left) and GalT I / (right) mice. (j,k) Sections of adrenal glands through their widest diameter (equator) illustrating the reduced size and stratification of the adrenal cortex. Most conspicuous is the dramatically reduced differentiation of the inner zona reticularis and intermediate zona fasciculata. All sections were stained with hematoxylin and eosin. Reprinted from Ref. [44].

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Fig. 3. Histological analysis (HE staining) of the skin of 10-day-old pups. (A and C) A wild-type (+/+) mouse; (B and D) a GalT I / mouse. Note that the prickle cell layer and granular layer are much thicker in the GalT I / mouse than in the normal mouse. Ep: epidermis. Original magnification:  100 (A and B),  400 (C and D). Reprinted from Ref. [45].

side-chains of glycoprotein hormones in GalT I-null animals should be associated with endocrine insufficiency, although the effects on specific oligosaccharide structures have not yet been determined. 4.4. Epithelial cells exhibit increased proliferation and abnormal differentiation Asano et al. [45] also reported a neonatal lethal phenotype for the GalT I-null animal. The age of lethality differed slightly from Lu et al., with only 50% of the GalT I-null animals dying during the neonatal period, another 30% dying as young adults (i.e., within 4 months), and 20% surviving to adulthood. Although an explanation for the lethality was not determined, the authors observed that GalT I-null animals exhibit an increased rate of cell proliferation in some epithelial cell types, relative to wild-type animals. The accelerated proliferation is detected in the stem cells of the epidermal basal layer of the skin and in the small intestine (Fig. 3). Cell proliferation in the skin leads to acanthosis and hyperkaeratosis of the young GalT I-null mice. In the intestine, cell proliferation results in enlarged intestinal crypts, and differentiation of intestinal villus cells appears abnormal as evidenced by analysis of the constituent disaccharidases. Lactase is localized over the entire surface of the villus epithelium in heterozygous mice but restricted to the top half of the villus in

GalT I-null mice. The villi of GalT I-null mice also exhibit precocious expression of maltase and sucrase – isomaltase relative to heterozygous mice. The authors conclude that GalT I reaction product, Galh1,4-GlcNAc, is critical for normal epithelial cell proliferation and differentiation. There are several factors that may account for the different interpretations of the GalT I-null neonatal lethal phenotype by Lu et al. [44] and Asano et al. [45]. The severity of the phenotype may be affected by the strain of mice used to create the null animal or protocols for handling the mice (light cycle, feed, etc.). The phenotypes may not be exclusive; rather, each group focused on different tissues when analyzing the mutant animals.

5. Null mutations in the long GalT I isoform Unlike that found in the total GalT I-null animal, the long GalT I-null animal exhibits near-normal levels of galactosylation as judged by two criteria. GalT I enzymatic activity toward GlcNAc substrates is 72% of wild-type levels in both spermatozoa and mammary glands [36,54]. Furthermore, glycoprotein galactosylation is indistinguishable between wild-type and GalT I-null testis as detected by RCA-I reactivity [36]. These experiments suggest that expression of the short GalT I isoform is sufficient to fulfill

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the biosynthetic function of GalT I in spermatozoa and mammary gland. Therefore, the phenotypes of the long GalT I-null animal are likely due to defects in non-biosynthetic functions of GalT I, such as its reported role as a cell surface receptor, rather than due to defects in galactosylation (Table 1). The long GalT I isoform has been implicated as a surface receptor for glycosides substrates in the extracellular matrix, particularly during sperm binding to the egg coat, and during cell interactions with the basal lamina. Consequently, the effects of selectively deleting the long GalT I isoform have been examined on fertilization and mammary gland morphogenesis. 5.1. Fertilization Species-specific gamete recognition occurs when the sperm binds to the extracellular matrix surrounding the egg, called the zona pellucida (ZP). The ZP contains three major families of glycoproteins: ZP1, ZP2 and ZP3 [55]. Sperm bind to the ZP by specifically interacting with the Olinked oligosaccharide chains on ZP3 [56]. This interaction requires a lectin-like receptor on the sperm plasma membrane. By several criteria, GalT I is the best candidate for the ZP3 receptor on sperm. GalT I is expressed on the plasma membrane overlying the acrosome where it behaves as an integral membrane glycoprotein [18,57,58]. GalT I-specific reagents competitively inhibit gamete recognition [18,57,59]. Sperm GalT I specifically interacts with terminal GlcNAc residues on the sperm-binding O-linked oligosaccharides of ZP3 [60]. Aggregation of GalT I, either with ZP3 or antibodies against recombinant GalT I, results in the induction of the pertussis toxin-sensitive acrosome reaction [61,62]. Selective overexpression of the long GalT I isoform on sperm leads to increased ZP3 binding, accelerated heterotrimeric G-protein activation and precocious acrosome reactions [21]. Finally, ectopic expression of the long GalT I isoform in Xenopus oocytes confers the ability to bind ZP3 and undergo heterotrimeric G-protein activation in response to GalT I aggregation [35]. The creation of GalT I-null mice allowed a more rigorous test of the involvement of sperm GalT I as a ZP3 receptor and mediator of the acrosome reaction [36]. Sperm from total GalT I-null males bind less radiolabeled ZP3 than wild-type sperm and are unable to undergo the acrosome reaction in response to either ZP3 or anti-GT antibodies, as do wild-type sperm. In contrast, GalT I-null sperm undergo the acrosome reaction normally in response to calcium ionophore, which bypasses the requirement for ZP3 binding. The inability of GalT I-null sperm to undergo a ZP3-induced acrosome reaction renders them physiologically inferior to wild-type sperm, as assayed by their relative inability to penetrate the egg coat and fertilize the oocyte in vitro. Sperm from both types of GalT I-null males, total and long isoform-null, demonstrate the same phenotype (Fig. 4). Therefore, since galactosylation during

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spermatogenesis appears normal in the long GalT I-null male, defects in ZP3 binding and induction of the acrosome reaction most likely reflect the loss of the long GalT I from the sperm surface, as opposed to defects in galactosylation [36]. Despite these defects, sperm from GalT I-null animals retain their ability to bind the ZP and GalT I-null males are fertile [36]. One hypothesis that may explain this unexpected fertility is that sperm-ZP binding is mediated by at least two receptor –ligand interactions. Although GalT I-ZP3 interaction may facilitate adhesion, its primary role appears to be to activate signaling pathways that lead to the acrosome reaction. A second, GalT I-ZP3independent interaction most likely mediates primary adhesion of the gametes. The fact that GalT I is expressed on the surface of all mammalian sperm assayed thus far [63 – 66], is consistent with it functioning as a nonspecies-specific signaling subunit of a larger multimeric gamete receptor complex. Conceptually, this paradigm is similar to the concerted action of selectins and integrins in lymphocyte adhesion to the endothelium [67]. We now have direct evidence that a ZP3-independent ligand is enriched in the post-ovulatory egg and mediates initial gamete adhesion [Rodeheffer and Shur, unpublished results]. 5.2. Mammary gland morphogenesis Development of the mammary gland is an example of a morphogenetic event thought to involve surface GalT I. It is well established that the short GalT I isoform performs an essential biosynthetic function in the lactating mammary gland. The steady-state levels of the short GalT I isoform increase during lactation, and it forms a heterodimer with a-lactalbumin, which increases the affinity of GalT I for glucose and allows for the synthesis of lactose [27,68]. A significant event in mammary gland morphogenesis is the elaboration and differentiation of the epithelial duct system, which is dependent upon proper interactions with the laminin-rich basal lamina [69,70]. Earlier in vitro studies had suggested that mammary epithelial cells use GalT I to facilitate their interactions with the basal lamina [71]. Transgenic mice that overexpress the long GalT I isoform were generated, and, not surprisingly, these females cannot support their litters due to an inability to lactate. Mammary glands from transgenic females are smaller, exhibit less complex branching, and terminate in smaller lobuloalveolae than wild-type glands. Consequently, the quantity of milk and milk-specific proteins (such as h-casein and whey acidic protein) is significantly decreased in mammary glands from pregnant transgenic females. When cultured on laminin-rich basement membrane, mammary epithelial cells from transgenic females fail to migrate and organize into milk-producing spheroids, as do cells from wild-type glands. These experiments reinforce the suggestion that

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Fig. 4. Phenotypes of GalT I-null sperm. Deletion of GalT I results in a loss of GalT from the sperm surface and an inability to bind the zona pellucida ligand, ZP3 (a). As a result, GalT I-null sperm are unable to undergo the acrosome reaction in response to either ZP3 or anti-GalT I antibodies (b), and leads to a relative inability to penetrate the zona pellucida in vitro (c). Similar phenotypes are observed when either both GalT I isoforms are eliminated, or when only the long isoform is eliminated, under which conditions galactosylation appears normal during spermatogenesis due to expression of the short isoform. Thus, the failure of sperm to bind ZP3, undergo acrosomal exocytosis, and penetrate the zona pellucida is a function of the long isoform, independent of its biosynthetic function. The short isoform can substitute for the biosynthetic function of the long isoform, but not its function as a ZP3 receptor on the sperm surface. Despite their inability to bind ZP3, GalT I-null sperm are still able to bind to the zona pellucida, although it does not lead to acrosomal exocytosis. Subsequent studies show that sperm – egg binding involves the sequential action of a GalT I-ZP3 independent receptor – ligand interaction, responsible for initial gamete adhesion, after which ZP3 binds to and aggregates GalT I, inducing the acrosome reaction. Surprisingly, GalT I-null males are fertile, suggesting that the reduced efficiency of zona penetration is sufficient for fertility in the absence of competing wild-type sperm.

mammary epithelial cells utilize surface GalT I to facilitate their interactions with the underlying basal lamina [70]. Based on these results, it was predicted that the mammary glands from mice lacking the long GalT I isoform would also exhibit abnormal ductal morphogenesis. In fact, these mice display a complementary phenotype to the transgenic mice that overexpress GalT I [54]. Mammary glands from long GalT I-null females do not express the long isoform, but GalT activity is 72% of wild-type levels, presumably due to the presence of the short GalT I isoform. In contrast, GalT activity on the surface of mammary epithelial cells is reduced to 40% of wild-type levels, suggesting that in the absence of the long isoform, the short

isoform can be expressed on the cell surface, albeit at reduced levels. As opposed to females that overexpress long GalT I, about 80% of females null for long GalT I are capable of lactating and supporting litters. The physiological basis for the remaining 20% is not understood. Mammary glands from prepubertal GalT I-null females exhibit increased branching morphogenesis and decreased interbranch distance. At pregnancy, this excessive branching results in a higher density of lobuloalveolae and increased numbers of spurious lobuloalveolae along the ducts, instead of the normal clustered pattern seen in wild-type mammary glands. In addition to being present in increased numbers, the lobuloalveolae

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undergo precocious differentiation and produce greater quantities of milk proteins than do wild-type glands [54]. One possible explanation for this result is that the integrity of the basal lamina in the GalT I-null mammary gland is compromised, allowing for more severe invasion of the ductal system. In support of this conclusion, laminin expression is decreased in the basal lamina of the GalT Inull mammary gland. The reduction of laminin is due to both decreased levels of laminin mRNA as well as elevated mRNA levels for the matrix metalloproteases MMP-7 and MT1-MMP. These results suggest that in the absence of surface GalT I, expression of matrix remodeling enzymes is increased and expression of laminin proteins is decreased, resulting in a modification of the basal lamina and abnormal branching morphogenesis [54].

6. What have we learned from the GalT I null mutations? 6.1. There are other gene products with GalT activity As discussed above, residual GalT activity and galactosylated products remain in the total GalT I-null animal. This predicts that other gene products must exist that encode previously unappreciated galactosyltransferase proteins. Using conserved sequence alignments as probes to search the database, three groups reported the identification and subsequent cloning of five additional GalT variants, all of which have been shown to transfer h1,4-galactose [72 – 76]. One experimental strategy that has been used recently to study the specificity of the GalT family members is to ectopically express these proteins in cells where they are not normally expressed and examine the resulting biosynthetic and signaling consequences. As discussed elsewhere, ectopically expressing the long isoform of GalT I in Xenopus oocytes leads to ZP3 binding and GalT I-dependent heterotrimeric G-protein activation [35]. Expression of GalT family members in Sf-9 cells, which have low levels of endogenous galactosyltransferase activity, demonstrate that GalT II, III, IV, V and VI can all galactosylate GlcNAc residues on N-linked oligosaccharides [77]. 6.2. GalT I is not essential for normal embryonic development, but is required for postnatal development Both groups reported that GalT I-null animals were born at near the expected numbers and with apparently normal features, although they were smaller than wild-type littermates. Presumably, the lack of major morphological defects implies that GalT I is not essential for normal embryonic development. However, between 10% and 20% of the expected GalT I-null animals are not accounted for at birth [44,45], indicating a poorly penetrant embryonic phenotype. Although the underlying cause of the embryonic lethality has not been addressed, recent experiments by Chen et al.

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[84] demonstrate that GalT I activity is required for Fringemediated inhibition of Jagged 1-induced Notch signaling. These results are discussed by R.S. Haltiwanger and P. Stanley in a separate review in this issue, and raise the intriguing possibility that the GalT I-dependent phenotype is attributed to a failure of Notch signaling during development. The relatively mild embryonic lethal phenotype may reflect the ability of other GalTs to compensate for the loss of GalT I. Elimination of a gene product often fails to produce the phenotype expected from in vitro studies. Other gene products (known or unknown) prove to have redundant and/or contributory roles that minimize the phenotype of the original knockout. There are many examples in which null mutations produce expected phenotypes in vitro but fail to produce any significant phenotype in vivo, including transcription factors [78], matrix receptors [79], cytoskeletal components [80], and intracellular signaling molecules [81]. 6.3. The two different GalT I isoforms have similar, but not identical functions There is abundant evidence to show that both GalT I isoforms function biosynthetically in the Golgi apparatus. However, there is also evidence to suggest that the long isoform can also serve as a signal transduction receptor for extracellular glycoside ligands. Selectively eliminating the long GalT I isoform in appropriately engineered mice confirms that the long isoform fulfills functions that cannot be compensated for by the short isoform. For example, long GalT I-null mice appear to have normal intracellular galactosylation, but sperm are unable to bind ZP3, undergo an acrosome reaction or penetrate the ZP, as do wild-type sperm. These results clearly illustrate the requirement of the long GalT I isoform for ZP3 binding and induction of the acrosome reaction [36]. Further support for this comes from the recent finding that expression of the long GalT I, but not the short GalT I, on Xenopus oocytes leads to ZP3 binding and pertussis toxin-sensitive heterotrimeric G-protein activation [35]. Similarly, elimination of the long GalT I isoform leads to defective mammary gland morphogenesis, although intracellular galactosyltransferase activity is near normal [54]. It is surprising that a relatively small amino acid extension on the cytoplasmic domain of GalT I can have significant consequences on its function. Whether this is due to specific amino acid residues that interact with chaperon proteins that enable GalT to reach the surface and associate with signal transduction cascades, or rather, is a consequence of altered conformation of the transmembrane domain that affects GalT I retention in the Golgi complex, is unknown at this time. Finally, it is important to emphasize that what appears to be mutually exclusive schools of thought regarding the functions of the long and short isoforms, in reality, are

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entirely compatible with one another. Functions attributed to the long GalT I cytoplasmic domain may very well be operational in the Golgi complex, as well as when GalT I is expressed on the cell surface. For example, heterotrimeric G-protein activation is thought to facilitate Golgi vesiculation during mitosis [82]. Conceivably, this Golgi-localized G-protein activation could be orchestrated by the long isoform of GalT I, similar to its ability to activate Gproteins on the sperm surface [62]. Similarly, the transcriptional and translational controls that are thought to be responsible for the selective increase in the short isoform during lactation [31 – 33], no doubt contribute to the tissuespecific expression of the two isoforms, independent of non-biosynthetic functions that may distinguish the two GalT I proteins. In any event, it is fair to say that targeted mutations in the GalT I gene have raised many intriguing questions regarding the function of the GalT family members, which will provide fruitful avenues of investigation for many years to come.

Acknowledgements Original work in the authors’ laboratory has been supported by grants from the National Institutes of Health. We are grateful to Brooke Elder for critical reading of the manuscript.

[10] [11] [12]

[13]

[14] [15]

[16]

[17]

[18]

[19]

[20]

References [1] I.P. Trayer, R.L. Hill, The purification and properties of the A protein of lactose synthetase, J. Biol. Chem. 246 (1971) 6666 – 6675. [2] N.L. Shaper, J.H. Shaper, J.L. Meuth, J.L. Fox, H. Chang, I.R. Kirsch, G.F. Hollis, Bovine galactosyltransferase: identification of a clone by direct immunological screening of a cDNA expression library, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 1573 – 1577. [3] H. Narimatsu, S. Sinha, K. Brew, H. Okayama, P.K. Qasba, Cloning and sequencing of cDNA of bovine N-acetylglucosamine h1,4galactosyltransferase, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 4720 – 4724. [4] H.E. Appert, T.J. Rutherford, G.E. Tarr, J.S. Wiest, N.R. Thomford, D.J. McCorquodale, Isolation of a cDNA coding for human galactosyltransferase, Biochem. Biophys. Res. Commun. 139 (1986) 163 – 168. [5] L.N. Gastinel, C. Cambillau, Y. Bourne, Crystal structures of the bovine h1,4-galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose, EMBO J. 18 (1999) 3546 – 3557. [6] B. Ramakrishnan, P.S. Shah, P.K. Qasba, a-Lactalbumin (LA) stimulates milk h1,4-galactosyltransferase I (h4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine: crystal structure of h4Gal-T1
LA complex with UDP-Glc, J. Biol. Chem. 276 (2001) 37665 – 37671. [7] L.C. Lopez, C.M. Maillet, K. Oleszkowicz, B.D. Shur, Cell surface and Golgi pools of h1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation, Mol. Cell. Biol. 9 (1989) 2370 – 2377. [8] L. Villevalois-Cam, K. Tahiri, G. Chauvet, B. Desbuquois, Insulininduced redistribution of the insulin-like growth factor II/mannose 6phosphate receptor in intact rat liver, J. Cell. Biochem. 77 (2000) 310 – 322. [9] J. Kawano, S. Ide, T. Oinuma, T. Suganuma, A protein-specific mono-

[21]

[22]

[23]

[24] [25]

[26]

[27]

[28]

clonal antibody to rat liver h1,4-galactosyltransferase and its application to immunohistochemistry, J. Histochem. Cytochem. 42 (1994) 363 – 369. B.D. Shur, S. Roth, Cell surface glycosyltransferases, Biochim. Biophys. Acta 415 (1975) 473 – 512. B.D. Shur, S. Evans, Q. Lu, Cell surface galactosyltransferase: current issues, Glycoconj. J. 15 (1998) 537 – 548. S. Roseman, The synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion, Chem. Phys. Lipids 5 (1970) 270 – 297. S. Roth, E.J. McGuire, S. Roseman, Evidence for cell surface glycosyltransferases: their potential role in cellular recognition, J. Cell Biol. 51 (1971) 536 – 547. B.D. Shur, Glycosyltransferases as cell adhesion molecules, Curr. Opin. Cell Biol. 5 (1993) 854 – 863. E.M. Bayna, J.H. Shaper, B.D. Shur, Temporally specific involvement of cell surface h1,4-galactosyltransferase during mouse embryo morula compaction, Cell 53 (1988) 145 – 157. J. Roth, E.G. Berger, Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae, J. Cell Biol. 93 (1982) 223 – 229. T.T. Nguyen, D.A. Hinton, B.D. Shur, Expressing murine h1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype, J. Biol. Chem. 269 (1994) 28000 – 28009. B.D. Shur, C.A. Neely, Plasma membrane association, purification, and partial characterization of mouse sperm h1,4-galactosyltransferase (published erratum appears in J. Biol. Chem. 1989 Mar 5;264(7): 4264), J. Biol. Chem. 263 (1988) 17706 – 17714. D.J. Taatjes, J. Roth, N.L. Shaper, J.H. Shaper, Immunocytochemical localization of h1,4galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies, Glycobiology 2 (1992) 579 – 589. T. Suganuma, H. Muramatsu, T. Muramatsu, K. Ihida, J. Kawano, F. Murata, Subcellular localization of N-acetylglucosaminide h1,4-galactosyltransferase revealed by immunoelectron microscopy, J. Histochem. Cytochem. 39 (1991) 299 – 309. A. Youakim, H.J. Hathaway, D.J. Miller, X. Gong, B.D. Shur, Overexpressing sperm surface h1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm – egg interactions, J. Cell Biol. 126 (1994) 1573 – 1583. B.E. Close, K.J. Colley, In vivo autopolysialylation and localization of the polysialyltransferases PST and STX, J. Biol. Chem. 273 (1998) 34586 – 34593. P.C. Marker, J.P. Stephan, J. Lee, L. Bald, J.P. Mather, G.R. Cunha, Fucosyltransferase I and H-type complex carbohydrates modulate epithelial cell proliferation during prostatic branching morphogenesis, Dev. Biol. 233 (2001) 95 – 108. L. Borsig, R. Kleene, A. Dinter, E.G. Berger, Immunodetection of a1,3-fucosyltransferase (FucT-V), Eur. J. Cell Biol. 70 (1996) 42 – 53. U. Mandel, H. Hassan, M.H. Therkildsen, J. Rygaard, M.H. Jakobsen, B.R. Juhl, E. Dabelsteen, H. Clausen, Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family, Glycobiology 9 (1999) 43 – 52. G.E. Bauer, J. Balsamo, J. Lilien, Cadherin-mediated adhesion in pancreatic islet cells is modulated by a cell surface N-acetylgalactosaminylphosphotransferase, J. Cell Sci. 103 (1992) 1235 – 1241. N.L. Shaper, G.F. Hollis, J.G. Douglas, I.R. Kirsch, J.H. Shaper, Characterization of the full length cDNA for murine h1,4-galactosyltransferase: novel features at the 5V-end predict two translation start sites at two in-frame AUGs, J. Biol. Chem. 263 (1988) 10420 – 10428. R.N. Russo, N.L. Shaper, J.H. Shaper, Bovine h1,4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains: in vitro translation experiments demonstrate that both the short and the long forms of the

C. Rodeheffer, B.D. Shur / Biochimica et Biophysica Acta 1573 (2002) 258–270

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

[46]

[47]

[48]

enzyme are type II membrane-bound glycoproteins, J. Biol. Chem. 265 (1990) 3324 – 3331. L. Mengle-Gaw, M.F. McCoy-Haman, D.C. Tiemeier, Genomic structure and expression of human h1,4-galactosyltransferase, Biochem. Biophys. Res. Commun. 176 (1991) 1269 – 1276. D.H. Joziasse, Mammalian glycosyltransferases: genomic organization and protein structure, Glycobiology 2 (1992) 271 – 277. A. Harduin-Lepers, J.H. Shaper, N.L. Shaper, Characterization of two cis-regulatory regions in the murine h1,4-galactosyltransferase gene: evidence for a negative regulatory element that controls initiation at the proximal site, J. Biol. Chem. 268 (1993) 14348 – 14359. B. Rajput, N.L. Shaper, J.H. Shaper, Transcriptional regulation of murine h1,4-galactosyltransferase in somatic cells: analysis of a gene that serves both a housekeeping and a mammary gland-specific function, J. Biol. Chem. 271 (1996) 5131 – 5142. M. Charron, J.H. Shaper, N.L. Shaper, The increased level of h1,4galactosyltransferase required for lactose biosynthesis is achieved in part by translational control, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14805 – 14810. S.C. Evans, A. Youakim, B.D. Shur, Biological consequences of targeting h1,4-galactosyltransferase to two different subcellular compartments, BioEssays 17 (1995) 261 – 268. X. Shi, S. Amindari, K. Paruchuru, D. Skalla, H. Burkin, B.D. Shur, D.J. Miller, Cell surface h1,4-galactosyltransferase-I activates G-protein-dependent exocytotic signaling, Development 128 (2001) 645 – 654. Q. Lu, B.D. Shur, Sperm from h1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly, Development 124 (1997) 4121 – 4131. T. Nilsson, J.M. Lucocq, D. Mackay, G. Warren, The membrane spanning domain of h1,4-galactosyltransferase specifies trans Golgi localization, EMBO J. 10 (1991) 3567 – 3575. R.D. Teasdale, G. D’Agostaro, P.A. Gleeson, The signal for Golgi retention of bovine h1,4-galactosyltransferase is in the transmembrane domain, J. Biol. Chem. 267 (1992) 4084 – 4096. A.S. Masibay, P.V. Balaji, E.E. Boeggeman, P.K. Qasba, Mutational analysis of the Golgi retention signal of bovine h1,4-galactosyltransferase, J. Biol. Chem. 268 (1993) 9908 – 9916. R.N. Russo, N.L. Shaper, D.J. Taatjes, J.H. Shaper, h1,4-Galactosyltransferase: a short NH2-terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for Golgi retention, J. Biol. Chem. 267 (1992) 9241 – 9247. N. Yamaguchi, M.N. Fukuda, Golgi retention mechanism of h1,4galactosyltransferase. Membrane-spanning domain-dependent homodimerization and association with a- and h-tubulins, J. Biol. Chem. 270 (1995) 12170 – 12176. K.J. Colley, Golgi localization of glycosyltransferases: more questions than answers, Glycobiology 7 (1997) 1 – 13. G.J. Strous, P. van Kerkhof, R.J. Fallon, A.L. Schwartz, Golgi galactosyltransferase contains serine-linked phosphate, Eur. J. Biochem. 169 (1987) 307 – 311. Q. Lu, P. Hasty, B.D. Shur, Targeted mutation in h1,4-galactosyltransferase leads to pituitary insufficiency and neonatal lethality, Dev. Biol. 181 (1997) 257 – 267. M. Asano, K. Furukawa, M. Kido, S. Matsumoto, Y. Umesaki, N. Kochibe, Y. Iwakura, Growth retardation and early death of h1,4galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells, EMBO J. 16 (1997) 1850 – 1857. M. Kido, M. Asano, Y. Iwakura, M. Ichinose, K. Miki, K. Furukawa, Presence of polysialic acid and HNK-1 carbohydrate on brain glycoproteins from h1,4-galactosyltransferase-knockout mice, Biochem. Biophys. Res. Commun. 245 (1998) 860 – 864. N. Nakamura, N. Yamakawa, T. Sato, H. Tojo, C. Tachi, K. Furukawa, Differential gene expression of h1,4-galactosyltransferases I, II and V during mouse brain development, J. Neurochem. 76 (2001) 29 – 38. M. Kido, M. Asano, Y. Iwakura, M. Ichinose, K. Miki, K. Furukawa,

[49] [50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67] [68]

[69]

269

Normal levels of serum glycoproteins maintained in h1,4-galactosyltransferase I-knockout mice, FEBS Lett. 464 (1999) 75 – 79. J.S. Axford, Glycosylation and rheumatic disease, Biochim. Biophys. Acta 1455 (1999) 219 – 229. T. Fulpius, F. Spertini, L. Reininger, S. Izui, Immunoglobulin heavy chain constant region determines the pathogenicity and the antigenbinding activity of rheumatoid factor, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 2345 – 2349. N. Kotani, M. Asano, Y. Iwakura, S. Takasaki, Impaired glactosylation of core 2 O-glycans in erythrocytes of h1,4-galactosyltransferase knockout mice, Biochem. Biophys. Res. Commun. 260 (1999) 94 – 98. J.U. Baenziger, E.D. Green, Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin, Biochim. Biophys. Acta 947 (1988) 287 – 306. N.R. Thotakura, D.L. Blithe, Glycoprotein hormones: glycobiology of gonadotrophins, thyrotrophin and free alpha subunit, Glycobiology 5 (1995) 3 – 10. K. Steffgen, K. Dufraux, H. Hathaway, Enhanced branching morphogenesis in mammary glands of mice lacking cell surface h1,4-galactosyltransferase, Dev. Biol. 224 (2002) 114 – 133. J.D. Bleil, P.M. Wassarman, Mammalian sperm – egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm, Cell 20 (1980) 873 – 882. H.M. Florman, P.M. Wassarman, O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity, Cell 41 (1985) 313 – 324. L.C. Lopez, E.M. Bayna, D. Litoff, N.L. Shaper, J.H. Shaper, B.D. Shur, Receptor function of mouse sperm surface galactosyltransferase during fertilization, J. Cell Biol. 101 (1985) 1501 – 1510. N.F. Scully, J.H. Shaper, B.D. Shur, Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation, Dev. Biol. 124 (1987) 111 – 124. B.D. Shur, N.G. Hall, A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida, J. Cell Biol. 95 (1982) 574 – 579. D.J. Miller, M.B. Macek, B.D. Shur, Complementarity between sperm surface h1,4-galactosyltransferase and egg-coat ZP3 mediates sperm – egg binding, Nature 357 (1992) 589 – 593. M.B. Macek, L.C. Lopez, B.D. Shur, Aggregation of h1,4-galactosyltransferase on mouse sperm induces the acrosome reaction, Dev. Biol. 147 (1991) 440 – 444. X. Gong, D.H. Dubois, D.J. Miller, B.D. Shur, Activation of a G protein complex by aggregation of h1,4-galactosyltransferase on the surface of sperm, Science 269 (1995) 1718 – 1721. R.A. Fayrer-Hosken, A.B. Caudle, B.D. Shur, Galactosyltransferase activity is restricted to the plasma membranes of equine and bovine sperm, Mol. Reprod. Dev. 28 (1991) 74 – 78. J.L. Larson, D.J. Miller, Sperm from a variety of mammalian species express h1,4-galactosyltransferase on their surface, Biol. Reprod. 57 (1997) 442 – 453. G. Huszar, M. Sbracia, L. Vigue, D.J. Miller, B.D. Shur, Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane h1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios, Biol. Reprod. 56 (1997) 1020 – 1024. M.W. Tengowski, M.J. Wassler, B.D. Shur, G. Schatten, Subcellular localization of h1,4-galactosyltransferase on bull sperm and its function during sperm – egg interactions, Mol. Reprod. Dev. 58 (2001) 236 – 244. L.A. Lasky, Selectins: interpreters of cell-specific carbohydrate information during inflammation, Science 258 (1992) 964 – 969. K. Brew, T.C. Vanaman, R.L. Hill, The role of a-lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction, Proc. Natl. Acad. Sci. U. S. A. 59 (1968) 491 – 497. G.B. Silberstein, Postnatal mammary gland morphogenesis, Microsc. Res. Tech. 52 (2001) 155 – 162.

270

C. Rodeheffer, B.D. Shur / Biochimica et Biophysica Acta 1573 (2002) 258–270

[70] H.J. Hathaway, B.D. Shur, Mammary gland morphogenesis is inhibited in transgenic mice that overexpress cell surface h1,4-galactosyltransferase, Development 122 (1996) 2859 – 2872. [71] M.H. Barcellos-Hoff, Mammary epithelial reorganization on extracellular matrix is mediated by cell surface galactosyltransferase, Exp. Cell Res. 201 (1992) 225 – 234. [72] N.W. Lo, J.H. Shaper, J. Pevsner, N.L. Shaper, The expanding h1,4galactosyltransferase gene family: messages from the databanks, Glycobiology 8 (1998) 517 – 526. [73] T. Sato, K. Furukawa, H. Bakker, D.H. Van den Eijnden, I. Van Die, Molecular cloning of a human cDNA encoding h1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc h1,4-galactosyltransferase, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 472 – 477. [74] R. Almeida, M. Amado, L. David, S.B. Levery, E.H. Holmes, G. Merkx, A.G. van Kessel, E. Rygaard, H. Hassan, E. Bennett, H. Clausen, A family of h4-galactosyltransferases: cloning and expression of two novel UDP-galactose:h-N-acetylglucosamine h1,4-galactosyltransferases, h4GalT2 and h4GalT3, J. Biol. Chem. 272 (1997) 31979 – 31991. [75] M. Amado, R. Almeida, T. Schwientek, H. Clausen, Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions, Biochim. Biophys. Acta 1473 (1999) 35 – 53. [76] K. Furukawa, T. Sato, h1,4-galactosylation of N-glycans is a complex process, Biochim. Biophys. Acta 1473 (1999) 54 – 66. [77] S. Guo, T. Sato, K. Shirane, K. Furukawa, Galactosylation of N-linked oligosaccharides by human h1,4-galactosyltransferases I, II, II, IV, V and VI expressed in Sf-9 cells, Glycobiology 11 (2001) 813 – 820.

[78] H. Weintraub, The MyoD family and myogenesis: redundancy, networks, and thresholds, Cell 75 (1993) 1241 – 1244. [79] H. Gardner, J. Kreidberg, V. Koteliansky, R. Jaenisch, Deletion of integrin a1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion, Dev. Biol. 175 (1996) 301 – 313. [80] Y. Doi, M. Itoh, S. Yonemura, S. Ishihara, H. Takano, T. Noda, S. Tsukita, Normal development of mice and unimpaired cell adhesion/ cell motility/actin-based cytoskeleton without compensatory upregulation of ezrin or radixin in moesin gene knockout, J. Biol. Chem. 274 (1999) 2315 – 2321. [81] D. Ilic, Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, T. Yamamoto, Reduced cell motility and enhanced focal adhesion contact formation in cells from FAKdeficient mice, Nature 377 (1995) 539 – 544. [82] C. Jamora, P.A. Takizawa, R.F. Zaarour, C. Denesvre, D.J. Faulkner, V. Malhotra, Regulation of Golgi structure through heterotrimeric Gproteins, Cell 91 (1997) 617 – 626. [83] R. Tozawa, S. Ishibashi, J. Osuga, K. Yamamoto, H. Yagyu, K. Ohashi, Y. Tamura, N. Yahagi, Y. Iizuka, H. Okazaki, K. Harada, T. Gotoda, H. Shimano, S. Kimura, R. Nagai, N. Yamada, Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit, J. Biol. Chem. 276 (2001) 12624 – 12628. [84] J. Chen, D.J. Moloney, P. Stanley, Fringe modulation of Jagged1induced Notch signaling requires the action of h4galactosyltransfer4galactosyltransferase-1, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13716 – 13721.