β4-Galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development

Glycobiology vol. 20 no. 10 pp. 1311–1322, 2010 doi: 10.1093/glycob/cwq098 Advance Access publication on June 23, 2010 β4-Galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development 2 2 3 Toshikazu Nishie , Yoko Hikimochi , Kota Zama , 2 2 2 Yoshiyasu Fukusumi , Mitutoshi Ito , Haruka Yokoyama , 2 3 1,2 Chie Naruse , Makoto Ito , and Masahide Asano 2 Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa 920-8640, Japan and 3Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka 812-8581, Japan Received on October 3, 2009; revised on June 16, 2010; accepted on June 17, 2010 Glycosphingolipids (GSLs) are important for various biological functions in the nervous system, the immune system, embryogenesis and in other tissues and processes. Lactosylceramide (LacCer), which is synthesized from glucosylceramide (GlcCer) by LacCer synthase, is a core structure of GSLs, including gangliosides. LacCer synthase was reported to be synthesized by the β4-galactosyltransferase-6 (β4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking β4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the β4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. β4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in β4GalT-5-deficient embryos and extraembryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when β4GalT-5 cDNA was introduced into β4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the β4GalT-5-deficient XEN cells. Hematoma and ectopically accumulated trophoblast giant cells were observed in the anti-mesometrial pole of the extra-embryonic tissues, although all three embryonic layers formed. β4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extraembryonic defects caused the early embryonic lethality. Our results suggest that β4GalT-5 is essential for extraembryonic development during early mouse embryogenesis. 1 To whom correspondence should be addressed: Tel.: +81-76-265-2460; Fax: +81-76-234-4240; e-mail: Keywords: galactosyltransferase / glycosphingolipid / lactosylceramide / tetraploid rescue / XEN cell Introduction Glycosphingolipids (GSLs) are involved in the dynamics of many cell membrane-associated events, including cellular interactions, signaling, and trafficking; they play pivotal roles in the nervous system, the immune system, embryogenesis, and in other tissues and processes (Wiegandt 1985). GSLs cluster with specific signal transducers, tetraspanins, adhesion receptors, and growth factor receptors to form integral components of plasma-membrane microdomains, especially glycosynaptic microdomains (Regina and Hakomori 2008). In particular, specific functional interactions between carbohydrate moieties on GSLs and bacterial toxins such as cholera toxin and botulinum toxin have been well studied (Yowler and Schengrund 2004). Recent progress in the studies of gene-engineered mice of glycosyltransferases and relevant enzymes involved in the synthesis and modification of GSLs has elucidated various biological functions of GSLs, especially in the nervous system (Furukawa et al. 2004). It is also reported that spermatogenesis is impaired in GM2/GD2 synthase-deficient mice because of the defect in the transportation of testosterone (Furukawa et al. 2004). The core structures of GSLs are glucosylceramide (GlcCer) and lactosylceramide (LacCer), and all gangliosides, GSLs containing sialic acid residues, are synthesized from them (Supplementary Figure 1). GlcCer is synthesized by GlcCer synthase via the transfer of glucose from UDP-glucose to ceramide, and LacCer is synthesized by LacCer synthase via the transfer of galactose from UDP-galactose to GlcCer. GlcCer synthase is encoded by the Ugcg gene (Ichikawa et al. 1996; Yamashita et al. 1999), but LacCer synthase is thought to be encoded by some of the six members of the β-1,4-galactosyltransferase (β4GalT) gene family, β4GalT-1 to -6. LacCer synthase was purified from rat brain homogenates, and the corresponding cDNA was identified as β4GalT-6 (Nomura et al. 1998). However, a mutant CHO cell line, Pro-5Lec20, which expresses neither the β4GalT-1 nor the β4GalT-6 mRNA, shows LacCer synthase activity and produces LacCer (Lee et al. 2001; Kolmakova and Chatterjee 2005). Since β4GalT-5 is most homologous to β4GalT-6, β4GalT-5 was another possible candidate for LacCer synthase, at least in CHO cells (Lee et al. 2001). Furthermore, LacCer synthase activity decreases about half of the control in human umbilical-vein endothelial cells transfected with siRNA against β4GalT-5 (Rajesh et al. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 1311 T Nishie et al. 2005). According to expression databases (Gene Atlas using BioGPS and NCBI's Gene Expression Omnibus), β4GalT-6, but not β4GalT-5, is strongly expressed in the adult brain, while the expression of β4GalT-5 is strongly increased during embryonic stem (ES) cell differentiation. This reciprocal expression pattern suggests the role of β4GalT-5 and β4GalT-6 in embryogenesis and the nervous system, respectively. It is, however, still unclear which β4GalT is responsible for the synthesis of LacCer in vivo. We have been studying the various biological roles of galactose-containing carbohydrates in animals by systematically generating and analyzing mice deficient in members of the β4GalT gene family. The β4GalTs transfer galactose (Gal) from UDP-Gal to a non-reducing terminal N-acetylglucosamine (GlcNAc) of N- and O-glycans to synthesize the Galβ1-4GlcNAc structure besides LacCer synthesis. The protein products of the β4GalT family share 25–55% amino acid sequence identity and are classified into three groups: β4GalT-1 and -2, β4GalT-3 and -4, and β4GalT-5 and -6 (Lo et al. 1998; Hennet 2002). Our studies on β4GalT-1-deficient (β4GalT1−/−) mice revealed that the glycans synthesized by β4GalT-1 play roles in epithelial cell growth and differentiation (Asano et al. 1997), inflammatory responses (Asano et al. 2003), skin wound healing (Mori et al. 2004), and IgA nephropathy development (Nishie et al. 2007). Another group reported that glycans synthesized by β4GalT-1 are involved in anterior pituitary hormone function and in fertilization (Lu et al. 1997; Lu and Shur 1997). We also showed that β4GalT-2−/− mice are impaired in spatial learning/memory and motor coordination/learning, with reduced expression of the HNK-1 carbohydrate in the brain (Yoshihar (...truncated)