Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice.

Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal -acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear.

A degron system targeting endogenous PD-1 inhibits the growth of tumor cells in mice.

Recently, targeted protein degradation systems have been developed using the ubiquitin-proteasome system. Here, we established Programmed cell death-1 (PD-1) knockdown mice as a model system for subjecting endogenous mouse proteins to the small molecule-assisted shutoff (SMASh) degron system. SMASh degron-tagged PD-1-mCherry in Jurkat cells and CD3 splenocytes were degraded by the NS3/4A protease inhibitors, asunaprevir (ASV) or grazoprevir (GRV).

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 β-1,4-galactosyltransferase-1 (β4GalT-1) could not support survival in mice exposed to a lethal dose of irradiation.

Characterization of Innate and Adaptive Immune Responses in PYNOD-Deficient Mice.

PYNOD (also called NLRP10) is a member of the nucleotide-binding domain and leucine-rich repeat containing family. Many members of this family play important roles in the activation and/or regulation of immune and inflammatory responses. We previously showed that PYNOD inhibits the IL-1β secretion in response to microbial infection in PYNOD-transgenic mice.

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

It is uncertain which β4-galactosyltransferase (β4GalT; gene name, B4galt), β4GalT-5 and/or β4GalT-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.

New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

Jmjd3 and Utx are demethylases specific for lysine 27 of histone H3. Previous reports indicate that Jmjd3 is essential for differentiation of various cell types, such as macrophages and epidermal cells in mice, whereas Utx is involved in cancer and developmental diseases in humans and mice, as well as regulation in zebrafish and nematodes. Here, we report that Jmjd3, but not Utx, is involved in axial skeletal formation in mice.

AP-1B-mediated protein sorting regulates polarity and proliferation of intestinal epithelial cells in mice.

Background & Aims: In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance.

EXTL2, a member of the EXT family of tumor suppressors, controls glycosaminoglycan biosynthesis in a xylose kinase-dependent manner.

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear.

Large-scale identification of target proteins of a glycosyltransferase isozyme by Lectin-IGOT-LC/MS, an LC/MS-based glycoproteomic approach.

Model organisms containing deletion or mutation in a glycosyltransferase-gene exhibit various physiological abnormalities, suggesting that specific glycan motifs on certain proteins play important roles in vivo. Identification of the target proteins of glycosyltransferase isozymes is the key to understand the roles of glycans. Here, we demonstrated the proteome-scale identification of the target proteins specific for a glycosyltransferase isozyme, β1,4-galactosyltransferase-I (β4GalT-I).

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function.

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly.

Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice.

Mutations in the key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosamine kinase, result in distal myopathy with rimmed vacuoles (DMRV)/hereditary inclusion body myopathy (HIBM) in humans. Sialic acid is an acidic monosaccharide that modifies non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, and it plays an important role in cellular adhesions and interactions. In this study, we generated mice with a V572L point mutation in the GNE kinase domain.

Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1.

Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1.

Methods: We first constructed octn1 gene knockout (octn1 ( -/- )) mice.

Impairment of embryonic cell division and glycosaminoglycan biosynthesis in glucuronyltransferase-I-deficient mice.

We have revealed that in Caenorhabditis elegans, non-sulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, whereas heparan sulfate is essential for embryonic morphogenesis in the later stages of development. To clarify the roles of chondroitin sulfate and heparan sulfate in early embryogenesis in mammals, we generated glucuronyltransferase-I (GlcAT-I) knock-out mice by gene targeting. GlcAT-I is an enzyme required for the synthesis of both chondroitin sulfate and heparan sulfate.

S1P(2), the G protein-coupled receptor for sphingosine-1-phosphate, negatively regulates tumor angiogenesis and tumor growth in vivo in mice.

Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the G(i)-coupled chemotactic receptor S1P(1). Here, we report that the distinct receptor S1P(2) is responsible for mediating the G(12/13)/Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P(2)(LacZ/+) mice, we found that S1P(2) was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC).

Important role of heparan sulfate in postnatal islet growth and insulin secretion.

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet beta-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery.

Learning/memory impairment and reduced expression of the HNK-1 carbohydrate in beta4-galactosyltransferase-II-deficient mice.

The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, alpha2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galbeta1-4GlcNAc and Galbeta1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in beta4-galactosyltransferase-II (beta4GalT-II).

Both alleles of PSF1 are required for maintenance of pool size of immature hematopoietic cells and acute bone marrow regeneration.

Hematopoietic stem cells (HSCs) have a very low rate of cell division in the steady state; however, under conditions of hematopoietic stress, these cells can begin to proliferate at high rates, differentiate into mature hematopoietic cells, and rapidly reconstitute ablated bone marrow (BM). Previously, we isolated a novel evolutionarily conserved DNA replication factor, PSF1 (partner of SLD5-1), from an HSC-specific cDNA library. In the steady state, PSF1 is expressed predominantly in CD34(+)KSL (c-kit(+)/Sca-1(+)/Lineage(-)) cells and progenitors, whereas high levels of PSF1 expression are induced in KSL cells after BM ablation.

The scaffold protein JSAP1 regulates proliferation and differentiation of cerebellar granule cell precursors by modulating JNK signaling.

Cerebellar granule cell precursors (GCPs) proliferate in the outer part of the external granular layer (EGL). They begin their differentiation by exiting the cell cycle and migrating into the inner part of the EGL. Here we report that JSAP1, a scaffold protein for JNK signaling pathways, is expressed predominantly in the post-mitotic GCPs of the inner EGL.

Ablation of the scaffold protein JLP causes reduced fertility in male mice.

The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the G(alpha13) and Cdo-mediated pathway, in vitro.

Neural-specific ablation of the scaffold protein JSAP1 in mice causes neonatal death.

We previously identified c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) as a scaffolding factor for JNK intracellular signaling pathways. Targeted gene-disruption studies have shown that JSAP1-null mice are unable to breathe and die shortly after birth. Although neural defects might be responsible for their death, there has been no convincing evidence for this.

Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain.

The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains.

PSF1 is essential for early embryogenesis in mice.

Psf1 (partner of sld five 1) forms a novel heterotetramer complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three, respectively, in Japanese), with Sld5, Psf2, and Psf3. The formation of this complex is essential for the initiation of DNA replication in yeast and Xenopus laevis egg extracts. Although all of the components are well conserved in higher eukaryotes, the biological function in vivo is largely unknown.

Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice.

Asparaginyl endopeptidase (AEP)/legumain, an asparagine-specific cysteine proteinase in animals, is an ortholog of plant vacuolar processing enzyme (VPE), which processes the exposed asparagine residues of various vacuolar proteins. In search for its physiological role in mammals, here we generated and characterized AEP-deficient mice. Although their body weights were significantly reduced, they were normally born and fertile.