The Lc3-synthase gene B3gnt5 is essential to pre-implantation development of the murine embryo

Abstract

Background: Glycosphingolipids (GSL) are integral components of mammalian cell membranes that are involved in cell adhesion and cell signaling processes. GSL are subdivided into structural series, like ganglio-, lacto/neolacto-, globo- and isoglo-series, which are defined by distinct trisaccharide cores. The beta1,3 N-acetylglucosaminyltransferase-V (B3gnt5) enzyme catalyzes the formation of the Lc3 structure, which is the core of lactoseries derived GSL.

Results: The biological significance of the glycoconjugates produced by the B3gnt5 enzyme was investigated by inactivating the B3gnt5 gene in the mouse germline. The disruption of the B3gnt5 protein-coding region in mouse embryonic stem cells resulted in reduced Lc3-synthase activity, supporting its specific contribution to lactoseries derived GSL synthesis. Breeding of heterozygous mutant mice failed to produce any viable progeny homozygous for the B3gnt5-null allele. The genotypic examination of embryos from heterozygous crosses showed that the disruption of the B3gnt5 gene leads to pre-implantation lethality. This finding was compatible with the expression pattern of the B3gnt5 gene in the pre-implantation embryo as shown by in situ hybridization. The analysis of GSL profiles in embryonic stem cells heterozygous for the B3gnt5-null allele confirmed the reduced levels of lactoseries derived GSL levels and of other GSL species.

Conclusion: The disruption of the B3gnt5 gene in mice affected the expression of lactoseries derived GLS and possibly of protein-bound beta3GlcNAc-linked glycans, thereby demonstrating an essential contribution of these glycoconjugates in early embryonic development, and supporting the importance of these glycoconjugates in cell differentiation and adhesion processes.

Figure 1

Biosynthesis of GSL core structures. GSL biosynthesis is initiated by the transfer of Glc to ceramide (Cer) catalyzed by the Ugcg enzyme. After addition of β1,4-linked Gal by Lac-Cer synthase, distinct core structures are defined by the action of different glycosyltransferases. The B3gnt5 enzyme catalyzes the Lc3 structure, which is the core of lactoseries GSL. The elongation of Lc3 by a α1,3-fucosyltransferase and a β1,4-galactosyltransferase yields the SSEA-1 antigen. ●, Glc; ○, Gal; ■, GlcNAc; ◆ Sia; ▲, Fuc.

Figure 2

B3gnt5 gene targeting in ES cells. A. The targeting vector was produced by cloning a neo resistance cassette into exon 4 of the mouse B3gnt5 gene. The construct was flanked by two genomic arms of 1.7 and 3.6 kbp. A genomic probe located just outside of the targeted region was used to detect homologous recombination. B. Genomic Southern blotting of ES cell clones. Genomic DNA of wildtype (WT) ES cells and of two ES cell clones bearing a homologously recombined B3gnt5 allele (5B5 and M20) were digested with EcoRI and hybridized to the genomic probe. C. Genotyping of wildtype (+) and targeted (-) B3gnt5 alleles by PCR as described in the Materials and Methods section.

Figure 3

N-acetylglucosaminyltransferase activity in ES cells. ES cells of the R1 and TC1 lines were lysed and N-acetylglucosaminyltransferase (GlcNAcT) activity was measured towards the acceptor substrates Gal(β-0)p-nitrophenol (pNP) and lactosylceramide (Lac-Cer). Black bars show GlcNAcT activity of wildtype ES cells and open bars show GlcNAcT activity of B3gnt5-targeted ES cells. Each value represents the average + S.D. of three independent measurements.

Figure 4

B3gnt5 expression in pre-implantation embryos. All pre-implantation stages investigated show the uniform presence of B3gnt5 transcripts up to the morula stage. B3gnt5 transcripts were confined to the inner cell mass at the blastocyst stage. Sense and antisense RNA probes were DIG-labeled as described in the Materials and Methods section. The pictures show embryos taken from C57Bl/6 wildtype matings.

Figure 5

Glycolipid analysis in ES cells. A. The oligosaccharide moiety of ES cell GSL was cleaved by ceramide glycanase, labeled with 2-aminobenzamide and separated by normal phase HPLC. The retention times of glucose units (GU) is indicated on the x-axis. The dotted line shows the GSL profile of wildtype R1 ES cells and the plain line shows the GSL profile of the R1 clone 5B5. B. Mass spectrometric fragmentation spectrum of the Lc6 oligosaccharide obtained from the corresponding HPLC fraction. The main fragment ions are labeled and their assignment to the Lc6 structure is depicted.