Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis

Abstract

Background: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts.

Results: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion.

Conclusions: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.

Figure 1

Lc3 synthase within the gangliosynthesis pathways. The X in the lacto-neolacto series biosynthesis pathway indicates a block due to disruption of the Lc3 synthase gene, B3gnt5. Six ganglioside synthesis pathways are shown: globo, gala, neolacto, lacto, a, and b. The molecules 3'-LM1, 3'-isoLM1, and 3',6'-isoLD1 are shown within the lacto-neolacto pathways. Gangliosides GM1 and GD1a are shown in the a-series pathway and ganglioside GD1b in the b-series pathway. Ganglioside nomenclature according to Svennerholm [44].

Figure 2

B3gnt5 gene knockout, conventional method. Conventional method for targeted disruption of B3gnt5. Given the genomic structure of the mutant allele, we designed primers spanning exon 4 and inserted a neo gene to distinguish all three genotypes. With exon 4 primers, knockout mice (-/-) displayed a nonspecific band at about 0.4 kb, as shown in lanes 1 and 2 of the PCR gel, with sequencing confirmation of nonspecific product. For heterozygous (+/-) and wild-type (+/+) mice, two bands were viewed at 0.4 kb and 0.5 kb. Sequence analysis of the 0.5-kb band confirmed that it was exon 4 of B3gnt5. With the inserted neo gene primer pair (N1R, N7R), both homozygous (-/-) and heterozygous (+/-) mice carried a positive 1.9-kb band, whereas a negative result in this PCR analysis indicated wild type (+/+).

Figure 3

B3gnt5 gene knockout, Cre-loxP method. Cre-loxP method for targeted disruption of B3gnt5. For the Cre-loxP model, two primer pairs were used also. The exon 4 primer pair was the same as that used for conventional knockout (Figure 2). Results were analyzed as for the conventional knockout method. Another primer set detected the inserted loxP site. Both homozygous (-/-) and heterozygous (+/-) mice carried a positive 2.1-kb band, which resulted from exon 4 deletion by Cre recombinase. A 4.3-kb band indicated when the targeted portion was not deleted by Cre recombinase. No band appeared in this PCR analysis for the wild-type mice (+/+). The PCR products were sequenced to further confirm the homogeneity.

Figure 4

B3gnt5 gene knockout analysis. A. Genomic Southern blot of ES cell clones. Genomic DNA from wild-type ES clones and possible candidate ES clones carrying homologously recombinant B3gnt5 allele were digested with the Xba I restriction enzyme and hybridized to the genomic probe. Two bands are shown, a 5.8-kb band of wild type and a 2.9-kb band (from Clones C10 and A5), indicating that the recombination occurred. B. Disruption of Lc3 synthase gene expression shown by RT-PCR. Disruption of Lc3 synthase gene expression shown by RT-PCR for the wild type (+/+), heterozygote (+/-), and homozygote (-/-). Total RNA was extracted from different organs in both adult and fetus and reversely transcripted into cDNA. PCR was performed by using cDNA as a template and primers located in exon 4 of Lc3 synthase. Beta actin was the internal quality control (right panels). a. Results from three different tissues in adult mice. In the wild-type (+/+) genotype, Lc3 synthase gene expression was detected mainly in spleen and was weakly positive in brain and liver. Lc3 synthase expression was decreased in the heterozygote (+/-) and completely knocked out in the homozygote (-/-). b. Results from head and body of E17 fetuses. Lc3 synthase gene expression was detected in both head and body tissue in the wild-type (+/+) fetus and the heterozygote (+/-) but was not detected in either head or body of the homozygote (-/-).

Figure 5

Early-stage growth delay of dwarfism in mice following Lc3 synthase knockout. The breeding was set in a heterozygote-heterozygote (+/-, +/-) base. Pups were weighed periodically. Mice of normal weight were weaned at day 21, and mice weighing <10 g were kept in the parental cage. The average weight of the same-litter control differs significantly from that of the dwarf B group (P = 0.0008), but differs less from that of the dwarf A group (P = 0.1072). Composition of the three mouse groups: Same-litter control (■): one +/+, two -/+, and one -/-; dwarf A group (□): two -/+; dwarf B group (●): two -/+.

Figure 6

Alopecia and obesity in Lc3 synthase knockout mice. A. Incidence of alopecia and obesity. A weight of >40 g in a male was counted as obesity. B. Alopecia. Fur loss was seen in all three genome types (wild-type data not shown) and could occur anywhere on the mouse's body. The arrowhead indicates where the fur loss caused difficulty in extension of hind limbs, and the arrows show fur growing again with less density. C. Late-stage obesity. Mice were weighed periodically. In panel b, the 24.6-g control with a genotype +/- was from the same litter as the obese mouse.

Figure 7

Spleen morphology of Lc3 synthase-knockout mice. Mouse spleens from three different genome types of similar age were sectioned and stained. The wild-type controls included a littermate (panel a) and a mouse from a non-knockout colony (panel b). White pulp is indicated by a white arrow in panel a, and the green arrow indicates red pulp. A germinal center is indicated by a white arrow in panel b. In both Wt/KO and KO/KO genotypes (panels c and d) tissue, the arrowhead indicates fused white pulp. The germinal center in the KO/KO mouse spleen (panel d) is not clearly defined.

Figure 8

Spleen immunophenotyping of Lc3 synthase-knockout mice. Mouse spleen immunophenotype analysis showing the fluorescence intensity of the staining of four antibodies: The intensity from T-cell antibodies (CD4 and CD8) was normal in the Wt/KO or KO/KO phenotype as compared to that in the Wt/Wt phenotype; that for NK cell antibody (CD49b) in the Wt/KO or KO/KO phenotype was similar to that of the Wt/Wt phenotype, with P ≥ 0.05; and that for B cells (CD19) in the Wt/KO or KO/KO phenotype was reduced by about 39% when compared to that of the Wt/Wt phenotype, with P < 0.001.

Figure 9

Anti-IgG response against 3'-isoLM1 and 3',6'-isoLD1. Four B3gnt5knockout mice, each shown as a separate curve, were immunized with purified gangliosides 3'-isoLM1 and 3',6'-isoLD1 that had been conjugated to Salmonella minnesota as a carrier and adjuvant. Sera from the immunized mice were tested by ELISA for reactivity against the purified gangliosides (3'-isoLM1 and 3',6'-isoLD1) as optical density readings at 492 nm (OD 492 nm).