Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase

Charlene A. Stern, Tamar R. Braverman and Michael Tiemeyer1

Department of Cell Biology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06510, USA

Received on June 1, 1999; revised on October 19, 1999; accepted on November 7, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide {alpha}2,3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.

Key words: ganglioside/Golgi apparatus/immunohisto­chemical localization/nervous system/sialyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The repertoire of oligosaccharides expressed at the cell surface reflects the regulated activity and expression of multiple protein families, including trimming enzymes, glycosyltransferases, substrate transporters and proteins directing Golgi and ER localization. Attempts to assess the relative contribution of glycosyltransferase regulation to the generation of glycan diversity have benefited from the molecular identification of new synthetic enzymes. In particular, biochemical purification, homology-based searches and expression cloning have successfully enlarged the described diversity of glycosyltransferase structure and function (Macher et al., 1991Go; Livingston and Paulson, 1993Go; Fukuda et al., 1996Go; Maly et al., 1996Go). As a result, molecular comparisons among transferases increasingly reveal structural motifs, frequently conserved across species, that impart acceptor/donor specificity or participate in catalysis (Datta and Paulson, 1995Go; Kapitonov and Yu, 1999Go). It is also apparent that while many transferases are evolutionarily well-conserved, some enzymatic activities have evolved independently in unrelated polypeptides (Lidholt et al., 1994Go; DeAngelis et al., 1997Go; Chen et al., 1999Go). Thus, despite the great differences evident in glycan structure across divergent species, distant phylogenetic comparisons of carbohydrate-directed binding and enzymatic activities have been informative.

We isolated a clone encoding the mouse sialyltransferase, mST3GalV (CMP-NeuAc: lactosylceramide {alpha}2,3-sialyltransferase), in a screen for a vertebrate homologue of Gliolectin, a Drosophila carbohydrate binding protein. mST3GalV synthesizes ganglioside GM3, the precursor for simple and complex a- and b-series gangliosides (van Echten and Sandoff, 1993Go; Ishii et al., 1998Go; Kono et al., 1998Go; Fukumoto et al., 1999Go). Therefore, the expression and regulated activity of mST3GalV/GM3 synthase is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in such diverse cellular processes as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell–cell interactions (Goldenring et al., 1985Go; Blackburn et al., 1986Go; Bremer et al., 1986Go; Kreutter et al., 1987Go; Nojiri et al., 1988Go; Tsuji et al., 1988Go; Hakomori and Igarishi, 1993Go, 1995; Yamamura et al., 1997Go). While greatly enriched in both neurons and glial cells, gangliosides are also found in non-neural tissue. Importantly, the first phenotypes described in transgenic knock-out mice lacking a key ganglioside synthetic enzyme, GM2/GD2 synthase, were manifest in non-neural tissue (Takamiya et al., 1998Go; Liu et al., 1999Go). Therefore, establishing the normal expression pattern of ganglioside biosynthetic enzymes should assist in evaluating future loss-of-function mutants.

The necessity for mST3GalV activity to act early in the pathway leading to synthesis of complex gangliosides has previously suggested that this enzyme should be localized to the cis-Golgi, analogous to the sequential topographical distribution of glycoprotein processing enzymes. However, characterization of the Golgi distribution of GM3 synthase activity by subcellular fractionation or in vitro pharmacologic manipulation has yielded reports of coenrichment with both proximal and distal Golgi markers (van Echten et al., 1990Go; Young et al., 1990Go; Iber et al., 1992Go; Lannert et al., 1998Go; Maccioni et al., 1999Go). Confocal immunohistochemical analysis of the subcellular localization of ganglioside biosynthetic enzymes, as we report here, provides an alternative to biochemical fractionation and allows spatial colocalization with defined Golgi markers in a minimally disrupted cell.

We have investigated the expression pattern and subcellular distribution of mST3GalV in neural and non-neural tissue. Immunohistochemistry and in situ hybridization reveal that mST3GalV is broadly expressed in neurons and glial cells throughout the nervous system. Robust expression of mST3GalV is evident in efferent neurons that project in myelinated pathways while oligodendrocytes express relatively low levels of the enzyme in comparison to neurons or Schwann cells. ST3GalV is highly expressed in distinct cell types in both liver and testis. Furthermore, the large and spatially distributed Golgi apparatus found in several neuronal types provides a favorable architecture for the colocalization of mST3GalV with markers that define Golgi subcompartments. By confocal analysis, mST3GalV fails to colocalize with a cis-Golgi marker but exhibits complete colocalization with a medial/trans-Golgi marker.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cloning of a cDNA encoding mouse ST3GalV
A nucleic acid hybridization screen was undertaken to isolate a vertebrate homologue of Gliolectin, a carbohydrate binding protein expressed in the embryonic nervous system of Drosophila melanogaster (Tiemeyer and Goodman, 1996Go). An embryonic mouse brain cDNA library was screened with a probe prepared from Drosophila Gliolectin coding sequence under conditions of moderate stringency. The sequence of a 1338 bp cDNA insert carried by a hybridizing phage clone revealed a 435 nucleotide (nt) open reading frame (ORF) at its 5'-end. The presence of a well-conserved, signature S-motif within the ORF identified the clone as a member of the sialyltransferase family. A combination of PCR and nucleic acid hybridization was employed to complete the full-length cDNA sequence which is identical to that recently identified as GM3 synthase, ST3GalV, GenBank Accession Number AB018048 (Ishii et al., 1998Go; Kono et al., 1998Go; Fukumoto et al., 1999Go).

Similarity between Drosophila Gliolectin, ST3GalV, and other sialyltransferases
Mouse ST3GalV possesses similarity to Drosophila Gliolectin across a 27 amino acid stretch that flanks the amino- and carboxy-terminal borders of the ST3GalV S-motif, displaying 26% amino acid identity and 59% similarity (81% weak similarity) (Figure 1A). Across this region, overall nucleotide identity between mST3GalV and Drosophila Gliolectin is 47% (excluding the S-motif) while the 25 nucleotide stretches 5' and 3' to the S-motif display, respectively, 64% and 72% identity between the two sequences. Upon optimal alignment, overall nucleotide identity between mST3GalV and Drosophila Gliolectin is 41%.



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Fig. 1. Mouse ST3GalV exhibits similarity to Drosophila Gliolectin and possesses a repeated motif flanking the S-motif. (A) The best alignment between mST3GalV and Drosophila Gliolectin identifies a short region of similarity that is continuous in Gliolectin but broken by the S-motif in mST3GalV. Backslashes indicate the position of S-motif residues (removed for clarity of presentation). (B) Within this region of mST3GalV, the amino acid sequence hDxxQxxxPxxxh is found repeated on either side of the S-motif. Among other sialyltransferases, this repeat is variably conserved. (C) In the {alpha}2–6 transferase, ST6GalI, repeat conservation is higher on the amino terminal side of the S-motif in mouse, human and chick. (D) Among {alpha}2–3 transferases, those utilizing N-acetyllactosamine acceptors exhibit greater repeat conservation on the carboxy-terminal side of the S-motif. (E) {alpha}2–3 transferases that transfer to Galß3GalNAc in mouse, human and chicken, possess a low level of repeat conservation. Sialyltransferase nomenclature is as described by Tsuji et al. (Tsuji et al., 1996Go). GenBank accession numbers for the aligned sequences are as follows: mST3GalV, AB018048; mST6GalI, BAA03680; hST6GalI, CAA35111; cST6GalI, CAA53235; mST3GalIII, CAA59013; hST3GalII, Q11203; mST3GalIV, CAA65076; hST3GalIV, Q11206; cST3, AAC14163; mST3GalI, CAA51919; hST3GalI, Q11201; cST3GalII, CAA56666; mST3GalII, CAA54294; m, mouse; h, human; c, chicken.

 
Within mST3GalV an amino acid sequence is repeated on either side of the S-motif (Figure 1B). Consisting of appropriately spaced amino acids, hDxxQxxPxxxh (where "h" denotes a hydrophobic, usually aromatic, residue), the repeat consensus is also conserved in other sialyltransferase family members. In particular, some enzymes possess greater repeat conservation on the amino-terminal (Figure 1C) or on the carboxy-terminal (Figure 1D) side of the S-motif while others demonstrate only weak similarity (Figure 1E). A similar repeat is not apparent in the mouse polysialyltransferase family (mST8SiaI-V) or in the chicken GalNAc {alpha}2,6 sialyltransferases (chST6GalNAcI/II).

Distribution of mST3GalV expression in neural cells
By in situ hybridization, mST3GalV mRNA was readily detectable in several populations of large, efferent neurons of the central nervous system (CNS). In particular, cerebellar Purkinje cells, spinal motorneurons and olfactory bulb mitral cells demonstrate significant mST3GalV mRNA expression (Figure 2). Some interneuron pools, such as spinal neurons within the dorsal horn, are also visualized by in situ hybridization. Consistent with the observed distribution of mRNA, antiserum (designated CS2) raised against an mST3GalV peptide (amino acids K227-I272) reveals an extensive and intricately arrayed Golgi apparatus within populations of large efferent neurons. Preimmune sera did not stain any of the tissues examined.



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Fig. 2. ST3GalV mRNA is detected in projection neurons of the central nervous system. Purkinje cells (Pu), the efferent cell population of the cerebellum, express high levels of mST3GalV message while hybridization to interneurons in the granule cell layer (Gr) and molecular cell layer (Mo) or to oligodendrocytes in the cerebellar white matter (WM) was below detection limits (A, anti-sense probe; B, sense probe). Similarly, motorneurons in the ventral horn of the spinal cord express high levels of mST3GalV message (C, anti-sense probe; D, sense probe). Scale bar corresponds to 50 µm in (A–D).

 
CS2 antiserum stains neurons in all regions of the nervous system. Within the cerebellar cortex, significant CS2 staining is associated with Purkinje cells, the sole output neurons of the cerebellum (Figure 3A,D). Immunohistochemical staining with CS2 antiserum is of sufficiently greater sensitivity compared to in situ hybridization that populations of smaller interneurons are also demonstrated to express mST3GalV. For instance, cerebellar granule cells and neurons within the cerebellar molecular layer contain CS2-labeled Golgi membranes. mST3GalV is also expressed in both pyramidal and nonpyramidal cell layers of neocortex (Figure 3G–H), and in ventral motorneurons and dorsal interneurons of the spinal cord, where stained membranes extend deeply into dendritic processes (Figure 3B,C,E,F). In the olfactory bulb, periglomerular, mitral, and granule cells express the enzyme (Figure 3I). Similarly, in hippocampus and dentate gyrus, both pyramidal and granule cells are stained with CS2 antiserum (data not shown). The strongest staining in the rostral neuraxis (mes-, di-, and telencephalic derivatives) was apparent in pyramidal cells of entorhinal cortex (Figure 3G). Generally, however, staining was greatest in large neurons situated in caudal structures such as the cerebellum, brainstem, and spinal cord (rhomb-, met-, and myelencephalic derivatives).



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Fig. 3. Immunohistochemical localization of mST3GalV in neural tissue with CS2 antiserum reveals elaborate, cell-specific Golgi architectural forms and differential expression among neural populations. (A, D) Cerebellar Purkinje cells (Pu) exhibit intense CS2 staining in large, perinuclear puncta (arrowhead in D). In granule cells (Gr), mST3GalV is restricted to a single locus in each cell (arrow in D). CS2 staining is also apparent in sparsely distributed cells of the molecular layer (Mo). The orientation of cerebellar layers, presented at low magnification in (A), is maintained at higher magnification in (D). (B, E) Motorneurons in the ventral horn of the spinal cord display an intricate, meshlike pattern of intracellular staining that extends for a considerable distance away from the nucleus but remains within the neuronal soma. A pool of ventral horn cells from the lumbar enlargement is shown at low magnification in (B) and a single motorneuron, also from the ventral horn of the lumbar spinal cord, is shown in (E). (C, F) In the dorsal horn of the spinal cord the intracellular distribution of mST3GalV correlates with cell location. The most superficial layers of the dorsal horn, presented toward the top in (C), contain cells that possess 1 to 2 puncta of CS2 staining (arrow in C). Cells in deeper layers of the dorsal horn exhibit a distributed pattern of CS2 staining, frequently seen to extend into at least one dendritic process (arrowhead in C and at higher magnification in F). (G–H) mST3GalV is distributed through all cortical layers in all regions examined although staining in entorhinal cortex (G) demonstrates the greatest intensity. A region of dorsolateral cortex midway between the frontal and occipital poles is presented in (H) for comparison. The pial surface (p) is indicated and the images extend to the depth of the corpus callosum (not shown) in (H) and to the lateral olfactory tract (not shown) in (G). (I) In the olfactory bulb, mST3GalV is present in periglomerular cells (arrow) surrounding all glomeruli (g), in the granule cell population (heavy arrow) and, most intensely, in mitral cells (arrowhead). Scale bar corresponds to 50 µm in A–C, 17 µm in D–F and 135 µm in G–I.

 
Expression of mST3GalV in CNS glial cells was significantly less apparent than expression in neurons. In regions of the CNS such as the corpus callosum or cerebellar peduncle, in which myelinating oligodendrocytes should constitute the primary cell population, only weak CS2 staining is detected (Figure 4A). Again, a rostral-to-caudal increase in expression was evident such that oligodendrocytes in spinal white matter were stained to a greater extent than were oligodendrocytes in the corpus callosum. In contrast, Schwann cells of the peripheral nervous system robustly express mST3GalV (Figure 4B).



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Fig. 4. Schwann cells express higher levels of mST3GalV than oligodendrocytes. (A) CS2 immunostaining of myelinated axon bundles in the cerebellar peduncle reveals only lightly stained intracellular structures (arrow) within oligodendrocytes. (B) In contrast, strong staining is evident in a compact, perinuclear compartment (arrow) in myelinating Schwann cells, here seen in a ventral nerve root that exited the spinal cord at a cervical level. Scale bar corresponds to 5 µm in both (A) and (B).

 
Distribution of mST3GalV expression in non-neural cells
In liver, sinusoidal endothelial cells stain with CS2 antiserum. Within a liver lobule, endothelial cell staining was restricted to a concentric ring of sinusoidal segments centered about the central vein while ST3GalV was not detectable in hepatocytes (Figure 5A–C). The unique sinusoidal staining pattern was reproducible in multiple liver lobules in each liver section from three adult mouse livers. Kupffer cells, the resident liver macrophage population, also express mST3GalV (Figure 5D). Identical results were seen with sections from three adult livers.



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Fig. 5. CS2 antiserum detects mST3GalV in the hepatic sinusoidal endothelium and in Kupffer cells. (A) anti-mST3GalV antibody stains a subset of sinusoids distributed broadly throughout the liver. (B) The sinusoids stained by CS2 antiserum form a ring, roughly concentric with the central vein (cv). (C) At higher magnification, ST3GalV expression is present in the endothelial cells lining sinusoids (s) but is not detected in surrounding hepatocytes. (D) Kupffer cells located both at the sinusoidal boundary and also within the liver parenchyma express mST3GalV. Scale bar corresponds to 475 µm in (A), 320 µm in (B), 5 µm in (C), 2.5 µm in (D).

 
In testis, both somatic and germ-line derivatives, including Leydig cells, Sertoli cells, spermatogonia, and primary spermatocytes, express ST3GalV (Figure 6A). Staining was not observed in matured spermatocytes consistent with previously described processes that modify and eliminate Golgi components during acrosome formation (Susi et al., 1971Go; Letts et al., 1974Go; Baccetti, 1975Go; Scully et al., 1987Go). Strong CS2 antiserum staining was present in the pseudostratified epithelial cells of the epididymis (Figure 6B,C).



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Fig. 6. ST3GalV is expressed in the male reproductive system. (A) CS2 staining is broadly distributed in cells of the seminiferous tubule, including germ cells (spermatogonia and primary spermatocytes, thin arrow) and non-germ cells (Sertoli, heavy arrow; Leydig cells, arrowhead) but is absent from cells in later stages of spermatogenesis (secondary spermatocytes, spermatids and spermatazoa). (B) Strong ST3GalV expression is observed in the pseudostratified epithelium lining the epididymis (L, epididymal lumen). (C) At higher magnification, the apical localization of CS2 staining in the epididymal epithelium is apparent (L, lumen; n, epithelial cell nucleus; arrow indicates the position of the basal lamina). Scale bar corresponds to 85 µm in (A), 45 µm in (B), and 2.5 µm in (C).

 
Anti-mST3GalV antisera recognizes a membrane protein in brain, liver, and testes
By Western blot analysis, CS2 antiserum recognizes a protein of apparent molecular weight 45 kDa in Golgi-enriched membrane preparations from brain, liver, and testes. The observed electrophoretic migration is consistent with the addition of between one and three N-linked oligosaccharides onto a polypeptide backbone of the mass predicted from the cDNA sequence, 41,263 Da (Figure 7). Preimmune sera did not exhibit reactivity.



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Fig. 7. Anti-mST3GalV antiserum, CS2, recognizes a single protein band by Western blot. A single polypeptide of apparent molecular weight 45 kDa is recognized by CS2 antiserum in Golgi-enriched membranes prepared from adult mouse brain (A), liver (B) or testes (C).

 
mST3GalV localizes to medial and trans-Golgi compartments in motorneurons
The sub-Golgi distribution of mST3GalV was investigated in spinal motorneurons by confocal immunofluorescent co-localization with multiple Golgi markers. Double immunostaining with CS2 antiserum and anti-GM130 antisera (Figure 8A) reveals that mST3GalV is not present in the cis-Golgi (Nakamura et al., 1995Go). However, mST3GalV completely colocalizes with a medial/trans-Golgi marker (Figure 8B), anti-Golgi antisera, which recognizes {alpha}-mannosidase II (Stieber et al., 1987Go; Rabouille et al., 1995Go). Our ability to discriminate Golgi subcompartments in spinal motorneurons is demonstrated by the lack of colocalization of GM130 and anti-Golgi antisera (Figure 8C). mST3GalV also partially colocalizes with Giantin (Figure 8D), a matrix protein broadly distributed across all Golgi compartments (Linstedt and Hauri, 1993Go; Sonnichsen et al., 1998Go). Identical results were seen in all neurons examined including cerebellar Purkinje and granule cells, brainstem neurons, and dorsal and ventral spinal neurons.



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Fig. 8. Confocal colocalization of mST3GalV demonstrates its presence in the medial/trans-Golgi of spinal motorneurons. (A) Double immunostaining with anti-GM130 (red) and CS2 antisera (green) reveals mST3GalV is not localized to the cis-Golgi. (B) Anti-Golgi antiserum (red), a medial/trans-Golgi marker, completely colocalizes with CS2 antiserum (green) yielding yellow fluorescence upon merge. (C) In spinal motorneurons, the cis-Golgi compartment, visualized with anti-GM130 (red), is well resolved from the medial/trans-Golgi, stained by anti-Golgi antiserum (green). (D) A broadly distributed Golgi matrix marker, anti-Giantin (red), partially colocalizes with CS2 antisera immunofluorescence (green). Scale bar corresponds to 20 µm in all panels.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In screening a mouse embryonic brain cDNA library for a vertebrate homologue of the Drosophila Gliolectin protein, we identified a mouse sialyltransferase, subsequently classified as mST3GalV (Tsuji et al., 1996Go). Lactosylceramide has been demonstrated to be a favored acceptor substrate for mST3GalV transferase activity, defining the enzyme as GM3 synthase (Ishii et al., 1998Go; Kono et al., 1998Go; Fukumoto et al., 1999Go). Consistent with the pivotal importance of GM3 to the biosynthesis of complex gangliosides, in situ hybridization and immunohistochemistry demonstrate that mST3GalV expression is broadly distributed in neural and non-neural tissue, although specific cell types can be grouped across a spectrum of staining intensities.

Amino acid alignment of Drosophila Gliolectin with mST3GalV reveals two regions of similarity, one on either side of the sialyltransferase S-motif. Each of these regions in mST3GalV is similar to the other, possessing a motif of appropriately spaced amino acids (hDxxQxxPxxxh). Drosophila Gliolectin, a type II transmembrane protein like mST3GalV, was originally identified based on its ability to bind a subset of GlcNAc-terminated glycosphingolipids isolated from Drosophila embryos (Tiemeyer and Goodman, 1996Go). Although mST3GalV and Drosophila Gliolectin exhibit divergent saccharide-binding specificities, (Galß4Glc- for ST3GalV and GlcNAcß3/4Hex- for Gliolectin) both proteins recognize carbohydrate in the context of a glycosphingolipid. In fact, mST3GalV appears to have at least 100-fold lower activity towards lactose than towards lactosylceramide (Kono et al., 1998Go). It has been suggested that the sialyltransferase S-motif may facilitate acceptor carbohydrate recognition (Datta and Paulson, 1995Go). Perhaps the extended S-motif consensus highlighted by alignment with Drosophila Gliolectin reflects the conservation of residues that accommodate glycosphingolipid within a binding site. Alternatively, this motif may reveal the signature of an ancestral carbohydrate recognition domain that has acquired new binding specificities relevant to the divergent glycan profiles of evolving organisms.

While Drosophila Gliolectin has not yet been demonstrated to possess enzymatic activity, the reaction catalyzed by ST3GalV defines the mouse sialyltransferase as a key component in the regulation of ganglioside biosynthesis. Synthesis of ganglioside GM3 is the first committed step in the formation of ganglioside. If vectorial elaboration, from cis- to trans-Golgi compartments, underlies ganglioside biosynthesis, the sub-Golgi localization of mST3GalV should identify the site where synthesis is initiated. Analysis of the sub-Golgi distribution of GM3 synthase activity by biochemical fractionation has indicated that the enzyme is enriched in the cis-compartment (Trinchera and Ghidoni, 1989Go; Trinchera et al., 1991Go; Iber et al., 1992Go). Consistent with this observation, pharmacologic manipulation of Golgi integrity by Brefeldin A, a fungal toxin that blocks intra-Golgi transport causing collapse of the Golgi (cis- through trans-compartments but not TGN) into the endoplasmic reticulum, has been reported to dissociate GM3 synthesis from later-acting ganglioside glycosyltransferases (van Echten et al., 1990Go; Young et al., 1990Go). However, other fractionation studies have demonstrated that synthesis of GM3 and its precursor (lactosylceramide) occurs in the trans-Golgi and TGN compartments where it is functionally coupled to other ganglioside glycosyltransferases (Lannert et al., 1998Go).

Reported differences in sub-Golgi localization of GM3 synthase activity may reflect Golgi organization that is specific to cell-type or cell maturity and may also be complicated by methodological considerations (Maccioni et al., 1999Go). Confocal immunohistochemical localization provides an alternate approach that allows assessment of enzyme distribution in a non-disrupted cell located in its normal tissue environment. Consistent with reports placing GM3 synthesis in later Golgi compartments, we find that anti-mST3GalV immunoreactivity fails to colocalize with a cis-Golgi marker, partially colocalizes with a broadly distributed Golgi marker and completely colocalizes with a medial/trans-Golgi marker in spinal motorneurons. The concentration of multiple ganglioside glycosyltransferases in distal Golgi compartments suggests that, compared to glycoprotein oligosaccharide processing, the control of ganglioside synthesis may be less reliant on topographical enzyme distribution or targeted vesicular traffic.

While all neural cell types (neuron and glia) synthesize ganglioside and should, therefore, express ST3GalV/GM3 synthase, determining the relative ganglioside content of neuronal or glial subsets is hampered by the difficulty inherent in obtaining appropriately enriched cell populations. However, cell-specific ganglioside expression has been demonstrated through analysis of glycolipids in mouse neurological mutants, in which defined neuronal populations are missing or degenerate in the perinatal period, and through biochemical characterization of ganglioside content in brain subcellular fractions (Suzuki et al., 1967Go; DeVries and Zmachinski, 1980Go; Seyfried et al., 1983Go; O'Gorman and Sidman, 1985Go). Immunohistochemical localization has provided another means of discerning the distribution of ganglioside expression (Kotani et al., 1993Go, 1994; Kotani et al., 1995Go). Anti-ganglioside antibodies reveal that a subset of the a- and b-series gangliosides, specifically GD1a and GT1b, are diffusely enriched throughout the adult rat cerebellar molecular layer, whose neuropil contains both the extensive Purkinje cell dendritic arbor and the parallel fibers of granule cells. In addition, GM1 and GM3 are enriched in cerebellar white matter which consists of myelinated Purkinje cell efferents and cerebellar afferent processes. Interestingly, however, the only characterized sialylated glycolipid reported to be enriched at the Purkinje cell somata, O-acetyl-LD1 (a disialylated neolactotetraosylceramide derivative), does not require the synthesis of GM3 as precursor. Therefore, Purkinje cells, which clearly express significant amounts of ST3GalV, demonstrate a robust ability to target the transport or sequestration of glycolipid to specific cell surface domains.

Immunohistochemical characterization of mST3GalV in mouse brain demonstrates that the enzyme is broadly but differentially expressed in neurons and glia. The common trait shared by neurons exhibiting robust anti-mST3GalV immunostaining is that their axonal projections form myelinated efferent pathways; projection lengths range from the relatively short pathway between cerebellar cortical Purkinje cells and cerebellar deep nuclei to the long-distance relay between ventral motor neurons and peripheral muscle. In contrast, small neurons, such as the granule cell populations of the cerebellar cortex, the olfactory bulb and the hippocampus, which give rise to non-myelinated local circuit projections, present lower levels of CS2 immunostaining. While differential staining between neuronal populations could arise from variations in Golgi architecture that generate alternate spatial distributions of a relatively constant amount of enzyme (Torre and Steward, 1996Go), the enrichment of mRNA for mST3GalV within large efferent neurons, revealed by in situ hybridization, is consistent with quantitative differences in enzyme expression.

Myelinated efferent projections that express high levels of ST3GalV differ from other neuronal pathways in at least one major respect. The interaction of the projecting neuron with an oligodendrocyte results in the formation of a myelin sheath. If axon caliber exceeds a threshold, neuron-glial communication commits the oligodendrocyte to myelination (Friede, 1972Go; Trapp et al., 1988Go). While ganglioside function in the initiation of myelination has yet to be conclusively demonstrated, axonal gangliosides recognized by Myelin Associated Glycoprotein, a member of the SIG-Lec family, contribute to the stabilization of formed myelin (Collins et al., 1997Go; Yin et al., 1998Go; Sheikh et al., 1999Go).

Although enriched in neural tissue, gangliosides are a normal constituent of non-neural cells as well. Previous analysis of hepatic ganglioside expression has shown that Kupffer cells and sinusoidal endothelial cells are enriched in gangliosides (primarily GM3 and other simple species) relative to hepatocytes, the only liver cells capable of expressing complex gangliosides (Gabellec et al., 1983Go; Senn et al., 1990Go). Furthermore, Kupffer cells and sinusoidal endothelial cells possess 7-fold higher GM3 synthase activity than hepatocytes (Gabellec et al., 1983Go; Senn et al., 1990Go). Consistent with the biochemical characterization of liver ganglioside expression, immunostaining for ST3GalV with CS2 antiserum is below detectable limits in hepatocytes but clearly evident in Kupffer cells and a spatially restricted subset of sinusoidal endothelial cells.

Hepatocytes and sinusoidal endothelial cells are born at the portal triad and progress during their life toward the central vein and senescence (Arber et al., 1988Go; Zajicek et al., 1988Go). Therefore, a centripetally graded increase in hepatocyte and non-parenchymal cell maturation exists along the hepatic cords. Furthermore, zonal subsegments of hepatic cords exhibit differential drug toxicity and pathophysiologic susceptibility that correlates with functional specialization (Jungermann and Katz, 1982Go; Tsukamoto et al., 1986Go). The distribution of ST3GalV suggests that regional differences in sinusoidal endothelial cells mirror hepatocyte specialization.

Analysis of mice lacking GM2/GD2 synthase, the enzyme acting after ST3GalV in the pathway from lactosylceramide to complex gangliosides, has implicated ganglioside function in spermatogenesis (Takamiya et al., 1998Go; Liu et al., 1999Go). In particular, testosterone accumulation in interstitial Leydig cells results in a decrease in circulating androgen levels in GM2/GD2 synthase null mice and a concomitant deficiency in germ cell maturation. The loss of complex ganglioside is accompanied by an increase in GM3 and GD3, primarily in non-germline cells, and altered Leydig cell morphology. Consistent with this reported shift in non-germline ganglioside distribution, we observe that ST3GalV immunoreactivity is high in Leydig cells. The reported absence of the stem cell population in some seminiferous tubules of GM2/GD2 synthase null mice and the expression of ST3GalV and GM2/GD2 synthase in a broad range of testicular cell types, including germ cells and epididymal epithelial cells, indicates that gangliosides function at multiple steps in spermatogenesis and spermiogenesis (Kawai et al., 1998Go).

With the cloning of ST3GalV by multiple approaches, at least one transferase has now been identified for each step in ganglioside oligosaccharide biosynthesis (Furukawa, 1998Go). Characterizing the wild-type distribution of synthetic enzymes is important for interpreting loss-of-function phenotypes arising from transgenic manipulation and will indicate which tissues may warrant closer examination. For instance, while global deletion of ST3GalV expression may be expected to present early-embryonic lethality, conditional loss-of-function in endothelial cells may reveal important aspects of liver organization while loss of ST3GalV expression in Purkinje cells would allow the assessment of ganglioside function in a myelinated efferent projection. Furthermore, the availability of appropriate reagents, targeting a complete ensemble of ganglioside biosynthetic enzymes, will reveal the subcellular spatial organization of glycosphingolipid synthesis and will allow evaluation of the topological interrelation between processing pathways for multiple glycoconjugate classes.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Lambda Zap mouse embryonic brain cDNA library, Lambda Fix II mouse genomic library, and ExAssist helper phage were obtained from Stratagene (La Jolla, CA). Trizol Reagent and Superscript II reverse transcriptase were supplied by GIBCO-BRL (Gaithersburg, MD). Biotinylated-poly(T) primer, streptavidin-magnetic beads, terminal deoxynucleotidyltransferase, Taq polymerase, digoxigenin-dUTP, and alkaline phosphatase–conjugated anti-digoxigenin Fab fragment were obtained from Boehringer Mannheim (Indianapolis, IN). Secondary antibodies (HRP-conjugated, alkaline phosphatase–conjugated, fluorescein-conjugated, and Texas red–conjugated goat anti-rabbit, Texas red–conjugated goat anti-Mouse, and unconjugated goat anti-rabbit IgG) were from Jackson Laboratories (West Grove, PA). pATH11 vector was from ATCC (Rockville, MD). TA cloning vector pCR2.1 was from Invitrogen (Carlsbad, CA). Affigel 15 resin was from Bio-Rad (Hercules, CA). ECL Plus Reagent was from Amersham (Arlington Heights, IL). Swiss Webster-CD1 mice were obtained from Charles River (Wilmington, MA). Antibody against GM130 was provided by G.Warren (Yale University, New Haven, CT), antibody against Giantin by G.Warren and H.Hauri (University of Basel, Switzerland) and anti-Golgi antisera by P.DeCamilli (Yale University). All other chemicals and reagents were obtained from standard commercial sources.

Cloning of mouse ST3GalV/GM3 synthase
Plaques (1 x 106) of a mouse embryonic brain cDNA library were screened at reduced stringency with a random-primed, 32P-dCTP labeled cDNA probe complementary to the full-length Drosophila Gliolectin coding sequence. Nylon filters bearing phage DNA were hybridized and washed at 55°C as described by Church and Gilbert (Church and Gilbert, 1984Go). Positive phage clones were plaque purified and inserts were recovered in Bluescript by in vivo excision with ExAssist helper phage. Nucleotide sequencing was performed by the dideoxy chain-termination method at the Keck Sequencing Facility (Yale University School of Medicine). The full-length sequence was subsequently completed by multiple approaches, including 5'RACE (Frohman, 1993Go), a genomic library screen (Lambda Fix II mouse genomic library) and RT-PCR (utilizing primers against Human EST AA386324). DNA sequence was obtained on both strands from at least three clones for the entire length of mST3GalV and was compiled, analyzed and aligned with the University of Wisconsin Genetics Computer Group software (GCG) and sequence analysis tools available through the National Center for Biotechnology Information (NCBI).

In situ hybridization
In situ hybridization was performed on 20 µm transverse sections of central nervous system regions taken from postnatal day 30 mice. Cyrostat sections were thaw mounted onto 3-aminopropyltriethoxysilane coated slides (2% AAS in acetone) and postfixed in 4% paraformaldehyde for 20 min. Solutions and subsequent treatment of slides were as previously described (Giger et al., 1996Go). Prehybridization and hybridization were performed in 0.1% w/v Ficoll, 0.1% w/v polyvinylpyrolidone, 0.1% bovine serum albumin, 0.025% w/v yeast tRNA, 0.02% w/v salmon sperm DNA, 50% formamide v/v in 5 x SSC. After prehybridization for 2 h at ambient temperature, the solution was removed and replaced with hybridization solution containing one-third of a T7 polymerase probe preparation (~50 ng of probe, see below). Following incubation at 55°C for 15 h in a humidified chamber, sections were washed sequentially in 5x SSC at 55°C for 5 min, 2x SSC at 55°C for 1 min, 1x SSC in 50% v/v formamide at 55°C for 30 min, and finally in 1x SSC at ambient temperature for 5 min. Hybridization of labeled probe was detected by incubation with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments diluted 1:5000 following the manufacturer’s recommended protocol. Alkaline phosphatase-induced chromophore precipitation (5-bromo-4-chloro-3-indolyl phosphate with nitroblue tetrazolium) was performed in the presence of 1 mM levamisole and tissue sections were subsequently dehydrated and mounted in Entellan.

To generate template for the incorporation of digoxigenin-UTP into RNA probes by T7 RNA polymerase, a 295 bp fragment of mST3GalV (from nt 727 to nt 1022) was generated by PCR and subcloned in both orientations into the TA cloning vector pCR2.1. A 1013 bp SpeI/AflIII fragment was then released from two plasmid clones containing insert in opposite orientation, sense or anti-sense relative to the T7 primer site. Following gel purification, 300 ng of fragment was used as template for probe synthesis as previously described (Giger et al., 1996Go). An aliquot of each T7 RNA polymerase product was spotted onto nylon membrane at multiple 2-fold dilutions and then probed with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments to verify that sense and anti-sense probe preparations achieved equivalent digoxigenin incorporation.

mST3GalV antibody production
An antibody to ST3GalV was raised against a TrpE fusion protein in rabbit. Nucleotides 709–847 (K227-I272) were subcloned into the pATH11 vector and expression of the fusion protein was induced in E.coli XL-1 Blue as described previously (Koerner et al., 1991Go). Fusion protein was purified by preparative SDS–PAGE followed by electroelution from the gel (Smith, 1992Go). To generate antigen for injection, the gel purified fusion protein was separated from excess free SDS by precipitation of the detergent as its potassium salt (Zaman and Verwilghen, 1979Go). One rabbit was initially injected with 200 µg protein of the resulting antigen preparation in complete Freund’s adjuvant and was subsequently boosted with the 100 µg of protein in incomplete Freund’s adjuvant every 3 weeks. The harvested serum was designated CS2 and a portion was affinity purified by binding to and eluting from an Affigel 15 column previously coupled with mST3GalV-TrpE fusion protein. All protein determinations were by the bicinchoninic acid assay (Smith et al., 1985Go).

Western blot analysis
Enriched Golgi membrane fractions were prepared from adult mouse brain, liver, and testis tissue as described previously (Fleischer and Kervina, 1974Go; Ma and Colley, 1996Go). Maximal recovery of immunoreactive mST3GalV required complete reduction and aggressive alkylation before SDS–PAGE; optimal reduction was determined to require 50 mM dithiothreitol followed by sulfhydral alkylation with 250 mM iodoacetamide. For SDS–PAGE, 1 µg membrane protein was electrophoresed and transferred to nitrocellulose (Harlow and Lane, 1988Go). Nitrocellulose blots were blocked in 5% dry milk, 0.2% Tween-20 in PBS. The primary antibody, anti-mST3GalV antisera (CS2 antisera), was diluted 1:5000 and the secondary antibody, HRP-conjugated goat anti-rabbit, was used at 1:5000 dilution. Antibody binding was detected by ECL Plus with the manufacturer’s recommended conditions. No difference was detected between blots probed with affinity-purified or nonpurified serum.

Immunohistochemistry and Immunofluorescence
Cortex, cerebellum, spinal cord, olfactory bulb, liver, and testis were harvested from three adult mice following perfusion with 4% paraformaldehyde in PBS. Dissected tissues were postfixed in 4% paraformaldehyde in PBS for an additional 4 h at 4°C and then equilibrated in sucrose solutions of increasing concentration to 18% final; 10 µm cyrostat sections were mounted onto Superfrost Plus slides (VWR) and stored dry at –20°C until use. Before immunostaining, slides were thawed to room temperature under dessication and the sections were then overlaid with wash buffer (0.3% Triton X-100, 0.45 M NaCl in 0.02 M Na2HPO4 pH 7.4). Tissue sections were blocked for 45 min in 15% normal goat serum, 0.3% Triton X-100, 0.3 M NaCl in 0.04 M Na2HPO4 pH 7.4. Primary antibody dilutions were 1:100 for CS2 and anti-Golgi antisera. Incubation with primary antisera was for 2 h in a humidified chamber at ambient temperature. Slides were then washed in Wash Buffer, reblocked for 15 min and incubated with secondary antibody (Goat anti-rabbit) conjugated either to HRP (1:500 dilution) or to alkaline phosphatase (1:1000 dilution). For HRP-conjugated secondary, antibody binding was visualized by precipitation of diaminobenzidine (DAB) in the presence of hydrogen peroxide. 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium were used as substrate for alkaline phosphatase conjugated secondaries. All slides were coverslipped under 50% glycerol in PBS. Immunohistochemical staining was identical in tissues probed with affinity-purified or nonpurified serum.

For immunofluorescent staining, primary antibody dilutions were 1:50 for CS2 and anti-GM130, 1:100 for anti-Giantin, and 1:30 for anti-Golgi antisera. Secondary antibodies, fluorescein-conjugated goat anti-rabbit, Texas-red goat anti-mouse, and Texas-red goat anti-rabbit, were used at a 1:250 dilution. Tissue sections were mounted in 4% w/v 1,4-diazabicyclo[2.2.2]octane (DABCO) in 50% glycerol in PBS and confocal images were acquired on a Zeiss Axiovert 100 microscope equipped with an argon/krypton laser. The microscope operates under control of the Zeiss LSM510 version 2.3 software package which was also used to acquire images. Optical sections were taken every 1.0 µm parallel to the coverslip. Acquired images were prepared for publication with Adobe software by minimally adjusting background fluorescence levels equally across all color channels. The relative fluorescence between red and green channels was unaltered from the original confocal scan.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dustin Khiem and Katherine Parisky for technical assistance in cDNA cloning, Graham Warren and Pietro DeCamilli for immunologic probes, and Tian Xu and Joseph Madri for their advice. This work was supported by grants from the NIH-NICHD (HD33878), by a Basil O’Connor Award from the March of Dimes and by The Patrick and Catherine Weldon Donaghue Foundation (all to M.T.). C.S. has received support from NIH-NIGMS (GM07223).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GM3, NeuAc{alpha}3Galß4GlcCer; GM2, GalNAcß4(Neu­Ac­{alpha}3)Gal­ß4GlcCer; GM1, Galß3GalNAcß4(NeuAc{alpha}3)Gal­ß4­GlcCer; GD2, GalNAcß4(NeuAc{alpha}8NeuAc{alpha}3)Galß4GlcCer; GD1a, NeuAc{alpha}3­Galß3GalNAcß4(NeuAc{alpha}3)Galß4GlcCer; GT1b, NeuAc{alpha}3Gal­ß3GalNAcß4(NeuAc{alpha}8NeuAc{alpha}3)Gal­ß4Glc­Cer; LD1, Galß3Glc­NAcß4(NeuAc{alpha}8NeuAc{alpha}3)Galß4­GlcCer; Cer, ceramide; ganglioside nomenclature is after Svennerholm (Svennerholm, 1964Go); sialyltransferase nomenclature is as described by Tsuji et al. (1996).


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Arber,N., Zajicek,G. and Ariel,I. (1988) The streaming liver II. Hepatocyte life history. Liver, 8, 80–87.[ISI][Medline]

Baccetti,B. (1975) The role of the Golgi complex during spermiogenesis. Curr. Topics Dev. Biol., 10, 103–122.[Medline]

Blackburn,C.C., Swank-Hill,P. and Schnaar,R.L. (1986) Gangliosides support neural retina cell adhesion. J. Biol. Chem., 261, 2873–2881.[Abstract/Free Full Text]

Bremer,E.G., Schlessinger,J. and Hakomori,S.-I. (1986) Ganglioside-mediated modulation of cell growth: specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J. Biol. Chem., 261, 2434–2440.[Abstract/Free Full Text]

Chen,S., Zhou,S., Sarkar,M., Spence,A.M. and Schachter,H. (1999) Expression of three Caenorhabditis elegans N-acetylglucosaminyltransferase I gene during development. J. Biol. Chem., 274, 288–297.[Abstract/Free Full Text]

Church,G.M. and Gilbert,W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA, 81, 1991–1995.[Abstract]

Collins,B.E., Kiso,M., Hasegawa,A., Tropak,M.B., Roder,J.C., Crocker,P.R. and Schnaar,R.L. (1997) Binding specificities of the sialoadhesin family of I-type lectins. Sialic acid linkage and substructure requirements for binding of myelin-associated glycoproetin, Schwann cell myelin protein and sialoadhesin. J. Biol. Chem., 272, 16889–16895.[Abstract/Free Full Text]

Datta,A.K. and Paulson,J.C. (1995) The sialyltransferase "sialylmotif" participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem., 270, 1497–1500.[Abstract/Free Full Text]

DeAngelis,P.L., Jing,W., Graves,M.V., Burbank,D.E. and Van Etten,J.L. (1997) Hyaluronan synthase of chlorella virus PBCV-1. Science, 278, 1800–1803.[Abstract/Free Full Text]

DeVries,G.H. and Zmachinski,C.J. (1980) The lipid composition of rat CNS axolemma-enriched fractions. J. Neurochem., 34, 424–430.[ISI][Medline]

Fleischer,S. and Kervina,M. (1974) Subcellular fractionation of rat liver. Methods Enzymol., 31, 6–40.[Medline]

Friede,R.L. (1972) Control of myelin formation by axon caliber (with a model of the control mechanism). J. Comp. Neurol., 144, 233–252.[ISI][Medline]

Frohman,M. (1993) Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol., 218, 340–356.[ISI][Medline]

Fukuda,M., Bierhuizen,M.F. and Nakayama,J. (1996) Expression cloning of glycosyltransferases. Glycobiology, 6, 683–689.[Abstract]

Fukumoto,S., Miyazaki,H., Goto,G., Urano,T., Furukawa,K. and Furukawa,K. (1999) Expression cloning of mouse cDNA of CMP-NeuAc:lactosylceramide a2,3-sialyltransferase and enzyme that initiates the synthesis of gangliosides. J. Biol. Chem., 274, 9271–9276.[Abstract/Free Full Text]

Furukawa,K. (1998) Recent progress in the analysis of ganglioside biosynthesis. Nagoya J. Med. Sci., 61, 27–35.[Medline]

Gabellec,M.M., Steffan,A.M., Dodeur,M., Durand,G., Kirn,A. and Rebel,G. (1983) Membrane lipids of hepatocytes, Kupffer cells and endothelial cells. Biochem. Biophys. Res. Commun., 113, 845–853.[ISI][Medline]

Giger,R.J., Wolfer,D.P., De Wit,G.M.J. and Verhaagen,J. (1996) Anatomy of rat Semaphorin III/Collapsin-1 mRNA expression and relationship to developing nerve tracts during neuroembryogenesis. J. Comp. Neurol., 375, 378–392.[ISI][Medline]

Goldenring,J.R., Otis,L.C., Yu,R.K. and Delorenzo,R.J. (1985) Calcium/gangliosdide-dependent protein kinase activity in rat brain membranes. J. Neurochem., 44, 1229–1234.[ISI][Medline]

Hakomori,S. and Igarashi,Y. (1995) Functional role of glycosphingolipids in cell recognition and signaling. J. Biochem., 118, 1091–1103.[Abstract]

Hakomori,S.-I. and Igarishi,Y. (1993) Gangliosides and glycosphingolipids as modulators of cell growth, adhesion and transmembrane signaling. Adv. Lipid Res., 25, 147–162.[ISI][Medline]

Harlow,E. and Lane,D. (1988) Immunoblotting. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 486–491.

Iber,H., van Echten,G. and Sandhoff,K. (1992) Fractionation of primary cultured cerebellar neurons: distribution of sialyltransferases involved in ganglioside biosynthesis. J. Neurochem., 58, 1533–1537.[ISI][Medline]

Ishii,A., Ohta,M., Watanabe,Y., Matsuda,K., Ishiyama,K., Sakoe,K., Nakamura,M., Inokuchi,J., Sanai,Y. and Saito,M. (1998) Expression cloning and functional characterization of human cDNA for ganglioside GM3 synthase. J. Biol. Chem., 273, 31652–31655.[Abstract/Free Full Text]

Jungermann,K. and Katz,N. (1982) Functional hepatocellular heterogeneity. Hepatology, 2, 385–395.[ISI][Medline]

Kapitonov,D. and Yu,R.K. (1999) Conserved domains of glycosyltransferases. Glycobiology, 9, 961–978.[Abstract/Free Full Text]

Kawai,H., Sango,K., Mullin,K. and Proia,R.L. (1998) Embryonic stem cells with distrupted GD3 synthase gene undergo neuronal differentiation in the absence of b-series gangliosides. J. Biol. Chem., 273, 19634–19638.[Abstract/Free Full Text]

Koerner,T.J., Hill,J.E., Myers,A.M. and Tzagoloff,A. (1991) High-expression vectors with multiple cloning sites for construction of trpE-fusion genes: pATH vectors. Methods Enzymol., 194, 477–490.[ISI][Medline]

Kono,M., Takashima,S., Liu,H., Inoue,M., Kojima,N., Young-Choon,L., Hamamoto,T. and Tsuji,S. (1998) Molecular cloning and functional expression of a fifth-type a2,3 sialyltransferase (mST3GalV: GM3 synthase). Biochem. Biophys. Res. Commun., 253, 170–175.[ISI][Medline]

Kotani,M., Kawashima,I., Ozawa,H., Terashima,T. and Tai,T. (1993) Differential distribution of major gangliosides in rat central nervous system detected by specific monoclonal antibodies. Glycobiology, 3, 137–146.[Abstract]

Kotani,M., Kawashima,I., Ozawa,H., Ogura,K., Ishizuka,I., Terashima,T. and Tai,T. (1994) Immunohistochemical localization of minor gangliosides in the rat central nervous system. Glycobiology, 4, 855–865.[Abstract]

Kotani,M., Terashima,T. and Tai,T. (1995) Developmental changes in ganglioside expression in postnatal rat cerebellar cortex. Brain Res., 700, 40–58.

Kreutter,D., Kim,J.Y.H., Goldenring,J.R., Rasmussen,H., Ukomadu,C., Delorenzo,R.J. and Yu,R.K. (1987) Regulation of protein kinase C activity by gangliosides. J. Biol. Chem., 262, 1633–1637.[Abstract/Free Full Text]

Lannert,H., Gorgas,K., Meibner,I., Wieland,F.T. and Jeckel,D. (1998) Functional organization of the Golgi apparatus in glycosphingolipid biosynthesis. J. Biol. Chem., 273, 2939–2946.[Abstract/Free Full Text]

Letts,P.J., Meistrich,M.L., Bruce,W.R. and Schachter,H. (1974) Glycoprotein glycosyltransferase levels during spermatogenesis in mice. Biochim. Biophys. Acta, 374, 192–207.

Lidholt,K., Fjelstad,M., Jann,K. and Lindahl,U. (1994) Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E.coli K5 capsular polysaccharides. Carbohydr. Res., 255, 87–101.[ISI][Medline]

Linstedt,A.D. and Hauri,H.-P. (1993) Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol. Biol. Cell, 4, 679–693.[Abstract]

Liu,Y., Wada,R., Kawai,H., Sango,K., Deng,C., Tai,T., McDonald,M.P., Araujo,K., Crawley,J.N., Bierfreund,U., Sandhoff,K., Suzuke,K. and Proia,R.L. (1999) A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder. J. Clin. Invest., 103, 497–505.[Abstract/Free Full Text]

Livingston,B.D. and Paulson,J.C. (1993) Polymerase chain reaction cloning of a developmentally regulated member of the sialyltransferase gene family. J. Biol. Chem., 268, 11504–11507.[Abstract/Free Full Text]

Ma,J. and Colley,K.J. (1996) A disulfide-bonded dimer of the Golgi ß-galactoside {alpha}2,6-sialyltransferase is catalytically inactive yet still retains the ability to bind galactose. J. Biol. Chem., 271, 7758–7766.[Abstract/Free Full Text]

Maccioni,H.J.F., Daniotti,J.L. and Martina,J.A. (1999) Organization of ganglioside synthesis in the Golgi apparatus. Biochim. Biophys. Acta, 1437, 101–118.[ISI][Medline]

Macher,B.A., Holmes,E.H., Swiedler,S.J., Stults,C.L.M. and Srnka,C.A. (1991) Human a1,3 fucosyltransferases. Glycobiology, 1, 577–584.[Medline]

Maly,P., Thall,A.D., Petryniak,B., Rogers,C.E., Smith,P.L., Marks,R.M., Kelly,R.J., Gersten,K.M., Cheng,G., Camphausen,R.T., Sullivan,F.X., Isogai,Y., Hindsgaul,O., von Andrian,U.H. and Lowe,J.B. (1996) The {alpha}(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis. Cell, 86, 643–653.[ISI][Medline]

Nakamura,N., Rabouille,C., Watson,R., Nilsson,T., Hui,N., Slusarewicz,P., Kreis,T.E. and Warren,G. (1995) Characterization of a cis-Golgi matrix protein, GM130. J. Cell. Biol., 131, 1715–1726.[Abstract]

Nojiri,H., Kitagawa,S., Nakamura,M., Kirito,K., Enomoto,Y. and Saito,M. (1988) Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60. J. Biol. Chem., 263, 7443–7446.[Abstract/Free Full Text]

O’Gorman,S. and Sidman,R.L. (1985) Degeneration of thalamic neurons in "Purkinje cell degeneration" mutant mice. I. Distribution of neuron loss. J. Comp. Neurol., 234, 277–297.[ISI][Medline]

Rabouille,C., Hui,N., Hunte,F., Kieckbusch,R., Berger,E.G., Warren,G. and Nilsson,T. (1995) Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci., 108, 1617–1627.[Abstract/Free Full Text]

Scully,N.F., Shaper,J.H. and Shur,B.D. (1987) Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. Dev. Biol., 124, 111–124.[ISI][Medline]

Senn,H.-J., Manke,C., Dieter,P., Tran-Thi,T.-A., Fijtzke,E., Gerok,W. and Decker,K. (1990) Ganglioside biosynthesis in rat liver: different distribution of gnaglioside synthases in hepatocytes, Kupffer cells and sinusoidal endothelial cells. Arch. Biochem. Biophys., 278, 161–167.[ISI][Medline]

Seyfried,T.N., Miyazawa,N. and Yu,R.K. (1983) Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant. J. Neurochem., 41, 491–505.[ISI][Medline]

Sheikh,K., Sun,J., Liu,Y., Kawai,H., Crawford,T.O., Proia,R.L., Griffin,J.D. and Schnaar,R.L. (1999) Mice lacking complex gangliosides develop Wallerian degenerations and myelination defects. Proc. Natl. Acad. Sci. USA, 96, 9532–9537.

Smith,J.A. (1992) In Ausubel,F.E., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K., editors. Electroelution of Protein from Stained Gels. New York: J Wiley. Chapter 10.5.

Smith,P.K., Krohn,R.I., Hermanson,G.T., Mallia,A.K., Gartner,F.H., Provenzano,M.D., Fujimoto,E.K., Goeke,N.M., Olson,B.J. and Klenk,D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 78–85.

Sonnichsen,B., Lowe,M., Levine,T., Jamsa,E., Dirac-Svejstrup,B. and Warren,G. (1998) A role for Giantin in docking COPI vesicles to Golgi membranes. J. Cell. Biol., 140, 1013–1021.[Abstract/Free Full Text]

Stieber,A., Gonatas,J.O., Gonatas,N.K. and Louvard,D. (1987) The Golgi apparatus-complex of neurons and astrocytes studied with and anti-organelle antibody. Brain Res., 408, 13–21.[ISI][Medline]

Susi,F.R., Leblond,C.P. and Clermont,Y. (1971) Changes in the Golgi apparatus during spermiogenesis in the rat. Am. J. Anat., 130, 251–268.[ISI][Medline]

Suzuki,K., Poduslo,S.E. and Norton,W.T. (1967) Gangliosides in the myelin fraction of developing rats. Biochim. Biophys. Acta, 144, 375–381.[ISI][Medline]

Svennerholm,L. (1964) The gangliosides. J. Lipid Res., 5, 145–155.[Abstract/Free Full Text]

Takamiya,K., Yamanoto,A., Furukawa,K., Zhao,J., Fukumoto,S., S,Y., Okada,M., Haraguchi,M., Shin,M., Kishikawa,M., Shiku,H., Aizawa,S. and Furukawa,K. (1998) Complex ganglioside are essential in spermatogenesis of mice: possible roles in the transport of testosterone. Proc. Natl. Acad. Sci. USA, 95, 12147–12152.[Abstract/Free Full Text]

Tiemeyer,M. and Goodman,C.S. (1996) Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system. Development, 122, 925–936.[Abstract/Free Full Text]

Torre,E.R. and Steward,O. (1996) Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture. J. Neurosci., 16, 5967–5978.[Abstract/Free Full Text]

Trapp,B.D., Hauer,P. and Lemke,G. (1988) Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells. J. Neurosci., 8, 3515–3521.[Abstract]

Trinchera,M. and Ghidoni,R. (1989) Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat Liver. J. Biol. Chem., 264, 15766–15769.[Abstract/Free Full Text]

Trinchera,M., Fabbri,M. and Ghidoni,R. (1991) Topography of glycosyltransferases involved in the initial glycosylations of gangliosides. J. Biol. Chem., 266, 20907–20912.[Abstract/Free Full Text]

Tsuji,A., Datta,A.K. and Paulson,J.C. (1996) Systematic nomenclature for sialyltransferases. Glycobiology, 6, v–xiv.[Medline]

Tsuji,S., Yamashita,T. and Nagai,Y. (1988) A novel carbohydrate signal-mediated cell surface protein phosphorylation: ganglioside GQ1b stimulates ecto-protein kinase activity on the cell surface of a human neuroblastoma cell line, GOTO. J. Biochem., 104, 498–503.[Abstract]

Tsukamoto,H., Towner,S., Ciofalo,L. and French,S. (1986) Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology, 6, 814–822.[ISI][Medline]

van Echten,G. and Sandoff,K. (1993) Ganglioside metabolism. J. Biol. Chem., 268, 5341–5344.[Free Full Text]

van Echten,G., Iber,H., Stotz,H., Takatsuki,A. and Sandoff,K. (1990) Uncoupling of ganglioside biosynthesis by brefeldin A. Eur. J. Cell Biol., 51, 135–139.[ISI][Medline]

Yamamura,S., Handa,K. and Hakomori,S. (1997) A close association of GM3 with c-src and rho in GM3-enriched microdomains at the B16 melanoma cell surface membrane: a preliminary note. Biochem. Biophys. Res. Commun., 236, 218–222.[ISI][Medline]

Yin,X., Crawford,T.O., Griffin,J.W., Tu,P., Lee,V.M., Li,C., Roder,J. and Trapp,B.D. (1998) Myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons. J. Neurosci., 18, 1953–1962.[Abstract/Free Full Text]

Young,W.W., Lutz,M.S., Mills,S.E. and Lechler-Osborn,S. (1990) Use of brefeldin A to define sites of glycosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to the brefeldin A block. Proc. Natl. Acad. Sci. USA, 87, 6838–6842.[Abstract]

Zajicek,G., Ariel,I. and Arber,N. (1988) The streaming liver III. Littoral cells accompany the streaming hepatocyte. Liver, 8, 213–218.[ISI][Medline]

Zaman,Z. and Verwilghen,R.L. (1979) Quantitation of proteins solubilized in sodium dodecyl sulfate-mercaptoethanol-tris electrophoresis buffer. Anal. Biochem., 100, 64–69.[ISI][Medline]