Kouichi Tachibana

MICAL, a Novel CasL Interacting Molecule, Associates with Vimentin

CasL/HEF1 belongs to the p130Cas family. It is tyrosine-phosphorylated following β1 integrin and/or T cell receptor stimulation and is thus considered to be important for immunological reactions. CasL has several structural motifs such as an SH3 domain and a substrate domain and interacts with many molecules through these motifs. To obtain more insights on the CasL-mediated signal transduction, we sought proteins that interact with the CasL SH3 domain by far Western screening, and we identified a novel human molecule, MICAL (a Molecule Interacting withCasL). MICAL is a protein of 118 kDa and is expressed in the thymus, lung, spleen, kidney, testis, and hematopoietic cells. MICAL has a calponin homology domain, a LIM domain, a putative leucine zipper motif, and a proline-rich PPKPP sequence. MICAL associates with CasL through this PPKPP sequence. MICAL is a cytoplasmic protein and colocalizes with CasL at the perinuclear area. Through the COOH-terminal region, MICAL also associates with vimentin that is a major component of intermediate filaments. Immunostaining revealed that MICAL localizes along with vimentin intermediate filaments. These results suggest that MICAL may be a cytoskeletal regulator that connects CasL to intermediate filaments.

Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15

A novel member of the human UDP‐N‐acetyl‐D‐galactosamine:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T) gene family was cloned as a homolog of human pp‐GalNAc‐T7, and designated pp‐GalNAc‐T10. pp‐GalNAc‐T10 transcript was found in the small intestine, stomach, pancreas, ovary, thyroid gland and spleen. In a polypeptide GalNAc‐transfer assay, recombinant pp‐GalNAc‐T10 transferred GalNAc onto a panel of mucin‐derived peptide substrates. Furthermore, pp‐GalNAc‐T10 demonstrated strong transferase activity with glycopeptide substrates.

Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, pp-GalNAc-T14☆

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) gene family was cloned and designated pp-GalNAc-T14. This type II membrane protein contains all motifs that are conserved in the pp-GalNAc-T family proteins and forms a subfamily with pp-GalNAc-T2 on the phylogenetic tree. Quantitative real time PCR analysis revealed significantly high expression of the pp-GalNAc-T14 transcript in kidney, although the transcripts were ubiquitously expressed in all tissues examined. Furthermore, the recombinant pp-GalNAc-T14 transferred GalNAc to a panel of mucin-derived peptide substrates such as Muc2, Muc5AC, Muc7, and Muc13 (-58). Our results provide evidence that pp-GalNAc-T14 is a new member of the pp-GalNAc-T family and suggest that pp-GalNAc-T14 may be involved in the O-glycosylation in kidney.

Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation

β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated β3GnT2-deficient (β3GnT2−/−) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in β3GnT2−/− mice. β3GnT2−/− T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3ε/CD28 and proliferated more strongly than T cells from WT mice. β3GnT2−/− B cells also showed hyperproliferation on BCR stimulation. Macrophages from β3GnT2−/− mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.

Molecular cloning and characterization of a novel member of the UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase family, pp‐GalNAc‐T121

We cloned in silico a novel human UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T), pp‐GalNAc‐T12. The deduced amino acid sequence of pp‐GalNAc‐T12 contains all conserved motifs in pp‐GalNAc‐T family proteins. Quantitative real time polymerase chain reaction analysis revealed that the pp‐GalNAc‐T12 transcript was expressed mainly in digestive organs such as stomach, small intestine and colon. The recombinant pp‐GalNAc‐T12 transferred GalNAc to the mucin‐derived peptides such as the Muc1a, Muc5AC, EA2 peptides and the GalNAc‐Muc5AC glycopeptide. Since mucins are glycoproteins mainly produced in the digestive organs, our results suggest that pp‐GalNAc‐T12 plays an important role in the initial step of mucin‐type oligosaccharide biosynthesis in digestive organs.

Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile.

A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis.

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

Significance Polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) belongs to the pp-GalNAc-T family, but its in vivo activity has not yet been identified. To investigate the functions of GALNTL5, we attempted to establish Galntl5-deficient mice and found that the heterozygous mutation of Galntl5 causes infertility in male mice because of immotile sperm. In these mice, glycolytic enzymes required for sperm motility were decreased, their protein loading into acrosomes was disrupted, and aberrant localization of the ubiquitin–proteasome system was observed. We found a patient diagnosed with asthenozoospermia, poor sperm motility, who had a mutation of the GALNTL5 gene in sperm and blood cells. Our data suggest that GALNTL5 is an essential functional molecule for sperm development, and the GALNTL5 mutation may cause human asthenozoospermia. For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin–proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

Regulation of cell shape and adhesion by CD34.

We previously reported that expression of CD43/leukosialin induces cell rounding and microvillus formation via inhibition of cell adhesion. Here, we found that CD34, a cell surface sialomucin and marker for hematopoietic progenitor cells, also inhibited cell adhesion and induced cell rounding and microvillus formation. Forced expression of CD34-induced cell rounding, microvillus formation, and phosphorylation of ezrin/radixin/moesin (ERM) proteins in HEK293T cells, while inhibiting integrin-mediated cell re-attachment. Furthermore, CD34+ blood cells and KG-1 cells, which express endogenous CD34 on their surface, were spherical in shape, surrounded by microvilli, and non-adherent to substrata. In addition, cleavage of O-sialomucin augmented integrin-mediated cell adhesion of KG-1 cells. These results suggest the involvement of CD34 in the inhibition of integrin-mediated cell adhesion and formation of the cell surface structure. The inhibitory function of CD34 in cell adhesion may affect cell shape organization via phosphorylation of ERM proteins. Cellular structures such as the spherical shape and microvilli of CD34+ cells may also contribute to regulation of cell adhesion.

Formation of microvilli and phosphorylation of ERM family proteins by CD43, a potent inhibitor for cell adhesion: Cell detachment is a potential cue for ERM phosphorylation and organization of cell morphology

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli, and phosphorylation of ERM in HEK293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HEK293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation, and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.

Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system

C1GalT and Cosmc bind by enzymatic study (View Interaction 1, 2, 3, 4)