Galactosyl carbohydrate residues on hematopoietic stem/progenitor cells are essential for homing and engraftment to the bone marrow.

The role of carbohydrate chains in leukocyte migration to inflamed sites during inflammation and trafficking to the lymph nodes under physiological conditions has been extensively characterized. Here, we report that carbohydrate chains also mediate the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM). In particular, we found that transplanted BM cells deficient in ß-1,4-galactosyltransferase-1 (ß4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation. BM cells obtained from mice deficient in ß4GalT-1 showed normal colony-forming activity and hematopoietic stem cell numbers. However, colony-forming cells were markedly rare in the BM of recipient mice 24 h after transplantation of ß4GalT-1-deficient BM cells, suggesting that ß4GalT-1 deficiency severely impairs homing. Similarly, BM cells with a point mutation in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene, encoding a key enzyme in sialic acid biosynthesis, showed mildly impaired homing and engraftment abilities. These results imply that the galactosyl, but not sialyl residues in glycoproteins, are essential for the homing and engraftment of HSPCs to the BM. These findings suggest the possibility of modifying carbohydrate structures on the surface of HSPCs to improve their homing and engraftment to the BM in clinical application.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Beta4-galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development.

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 beta4-galactosyltransferase-6 (beta4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking beta4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the beta4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. beta4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in beta4GalT-5-deficient embryos and extra-embryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when beta4GalT-5 cDNA was introduced into beta4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the beta4GalT-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. beta4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extra-embryonic defects caused the early embryonic lethality. Our results suggest that beta4GalT-5 is essential for extra-embryonic development during early mouse embryogenesis.

Development of IgA nephropathy-like disease with high serum IgA levels and increased proportion of polymeric IgA in Beta-1,4-galactosyltransferase-deficient mice.

The glycosylation of glycoproteins is important for their biological activity, conformation and stability. Recent studies indicate that aberrant glycosylation causes various human disorders. Here we report that mice lacking beta-1,4-galactosyltransferase-I (beta4GalT-I), which transfers galactose from UDP-Gal to terminal GlcNAc of N- and O-glycans in a beta-1,4- linkage, developed IgA nephropathy (IgAN)-like disease. Urinary albumin levels were significantly increased in the beta4GalT-I-deficient mice. Hematuria was detected in some of the beta4GalT-I-deficient mice, suggesting impaired renal function. Furthermore, histological and immunohistochemical examination showed expanded mesangial matrix, IgA deposition with mesangial pattern and electron-dense deposits in the paramesangial regions in the beta4GalT-Ideficient mice. These results demonstrate that the beta4GalT-I-deficient mice developed IgANlike disease. Furthermore, high serum IgA levels with increased polymeric forms were detected. In humans, serum IgA derived from patients with IgAN has aberrant beta3-galactosylation and sialylation on its O-linked glycans of the hinge region. Mouse IgA does not have O-glycans of the hinge region and has several N-glycans. As expected, beta4-galactosylation on the N-glycans of the serum IgA of the beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We suggest that aberrant beta4-galactosylation of serum IgA participates in the Nishie/Miyaishi/Azuma/Kameyama/Naruse/Hashimoto/Yokoyama/Narimatsu/Wada/Asano 126 development of IgAN, including deposition of IgA, polymerization of IgA, and glomerular injury after IgA deposition.

Development of immunoglobulin A nephropathy- like disease in beta-1,4-galactosyltransferase-I-deficient mice.

Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.

Functional conservation between fission yeast moc1/sds23 and its two orthologs, budding yeast SDS23 and SDS24, and phenotypic differences in their disruptants.

The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Deltamoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Deltasds23 Deltasds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar.

Impairment of skin wound healing in beta-1,4-galactosyltransferase-deficient mice with reduced leukocyte recruitment.

Cell-surface carbohydrate chains are known to contribute to cell migration, interactions, and proliferation, but their roles in skin wound healing have not been evaluated. We examined the biological roles of beta4-galactosylated carbohydrate chains in skin wound healing using mutant mice that lack beta-1,4-galactosyltransferase-I (beta4GalT-I), which is responsible for the biosynthesis of the type 2 chain in N-glycans and the core 2 branch in O-glycans. beta4GalT-I-deficient mice showed significantly delayed wound healing with reduced re-epithelialization, collagen synthesis, and angiogenesis, compared with control mice. Neutrophil and macrophage recruitment at wound sites was also impaired in these mice probably because of selectin-ligand deficiency. In accordance with the reduced leukocyte infiltration, the expression levels of macrophage-derived chemokines, transforming growth factor-beta1, and vascular endothelial growth factor were all reduced in beta4GalT-I(-/-) mice. These results demonstrate that beta4-galactosylated carbohydrate chains play a critical role in skin wound healing by mediating leukocyte infiltration and epidermal cell growth, which affects the production of chemokines and growth factors. This study introduces a suitable mouse model for investigating the molecular mechanisms of skin wound healing and is the first report showing that carbohydrate chains have a strong influence on skin wound healing.