The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

A neural glycotope, human natural killer-1 carbohydrate, is involved in synaptic plasticity. The key biosynthetic enzyme is a glucuronyltransferase, GlcAT-P, a type II membrane protein comprising an N-terminal cytoplasmic tail, transmembrane domain, stem region, and C-terminal catalytic domain. Previously, we reported that GlcAT-P has two isoforms differing in only the presence or absence of the N-terminal 13 amino acids (P-N13) in the cytoplasmic tail, but the functional distinction of these two isoforms has not been reported. Herein, we show that when expressed in Neuro2A cells, short form GlcAT-P (sGlcAT-P) exhibited significantly higher glucuronylation activity than the longer form (lGlcAT-P), despite their comparable specific activities in vitro. In addition, sGlcAT-P was strictly localized in Golgi apparatus, whereas lGlcAT-P was mainly localized in Golgi but partly in the endoplasmic reticulum. We demonstrated that the small GTPase, Sar1, recognized a dibasic motif in the cytoplasmic tail near P-N13 that was important for exiting the endoplasmic reticulum, and Sar1 interacted with sGlcAT-P more strongly than lGlcAT-P. Finally, the attachment of P-N13 to another glycosyltransferase, polysialyltransferase-I (ST8Sia-IV), had similar effects, such as reduced activity and entrapment within endoplasmic reticulum. These results suggest that P-N13 can control glycosyltransferase transport through Sar1 binding interference.

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

Glycans are known to play important roles in vertebrate development; however, it is difficult to analyze in mammals because it takes place in utero. Therefore, we used medaka (Oryzias latipes) to clarify the roles of glycans during vertebrate development. beta-1,4-Galactosyltransferase is one of the key enzymes in the biosynthesis of the lactosamine structures that are commonly found on glycoproteins and glycolipids. Here, we show the essential role of beta4GalT2 during medaka development. Depletion of beta4GalT2 by morpholino antisense oligonucleotide injection resulted in significant morphological defects, such as shortening of the anterior-posterior axis, cyclopia, impaired somite segmentation, and head hypoplasia. In situ hybridization analyses revealed that the loss of beta4GalT2 led to defective anterior-posterior axis elongation during gastrulation without affecting organizer formation. Furthermore, a cell tracing experiment demonstrated that beta4GalT2 knockdown mainly affects mediolateral cell intercalation, which contributes to anterior-posterior axis elongation. A cell transplantation experiment indicated that glycans are produced by beta4GalT2 cell-autonomously during gastrulation. beta4GalT2 depletion also led to enhanced apoptosis; however, this does not account for the phenotypic abnormalities, as blockade of apoptosis failed to compensate for the beta4GalT2 depletion. Our data suggest that beta4GalT2 activity is cell-autonomously required in cells undergoing mediolateral cell intercalation, which drives extension movements during medaka gastrulation.