Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan

Glycoproteins are decorated with complex glycans for protein functions. However, regulation mechanisms of complex glycan biosynthesis are largely unclear. Here we found that bisecting GlcNAc, a branching sugar residue in N-glycan, suppresses the biosynthesis of various types of terminal epitopes in N-glycans, including fucose, sialic acid and human natural killer-1. Expression of these epitopes in N-glycan was elevated in mice lacking the biosynthetic enzyme of bisecting GlcNAc, GnT-III, and was conversely suppressed by GnT-III overexpression in cells. Many glycosyltransferases for N-glycan terminals were revealed to prefer a nonbisected N-glycan as a substrate to its bisected counterpart, whereas no up-regulation of their mRNAs was found. This indicates that the elevated expression of the terminal N-glycan epitopes in GnT-III-deficient mice is attributed to the substrate specificity of the biosynthetic enzymes. Molecular dynamics simulations further confirmed that nonbisected glycans were preferentially accepted by those glycosyltransferases. These findings unveil a new regulation mechanism of protein N-glycosylation.This work was supported by Grant-in-Aid for Scientific Research (C) to Y.K. [17K07356], Leading Initiative for Excellent Young Researchers (LEADER) project to Y.K. from the Japan Society for the Promotion of Science (JSPS), by Takeda Science Foundation, and by Mochida Memorial Foundation for Medical and Pharmaceutical Research

N-acetylglucosaminyltransferase-V (GnT-V)-enriched small extracellular vesicles mediate N-glycan remodeling in recipient cells

Summary: Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs’ role in glycan remodeling in cancer metastasis

NAMIHEI: A Novel Algorithm for Genomic Polymorphism Detection from DNA Sequence

Introduction Potential genomic polymorphisms such as single nucleotide polymorphisms (SNPs) and insertions/ deletions (indels) have been discovered from a multiple alignment of DNA sequences. However, most of conventional alignment algorithms are originally designed for assembling fragment sequences into contigs, and usually are not suitable for detecting genomic polymorphisms. For example, it is almost impossible to detect polymorphisms on the traces are much noisy and the downstream region of a heterozygous indel where maternal or paternal traces shift in some distance. In our previous work, we reported a unique computer algorithm called Jessica for detecting indels from DNA sequences [1]. In this work, we developed another algorithm named NAMIHEI for discovering and genotyping of both SNPs and indels, making use of some features hidden in traces and a public database sequence as a standard reference. Furthermore, we also present an application program equipped with an e#cient use

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box.

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes-usually used for molecular analysis-were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine

Full-term development of embryos cultured in the warm box.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in the warm box.</p

Full-term development of inbred or outbred mouse strain embryos transported by parcel-delivery service.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of inbred or outbred mouse strain embryos transported by parcel-delivery service.</p

The rates of blastocyst development from zygotes cultured in three different types of microtubes with or without washing.

<p>Rates of development to the morula/blastocyst stages were examined at 72 h after insemination. Key: +, microtubes were washed with KSOM before embryo culture; –, microtubes were used without washing; dish, embryos were cultured in a culture dish by conventional methods as controls. *<i>P</i> < 0.05 by χ² test.</p

Full-term development of BDF1 mouse embryos transported by parcel-delivery service.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of BDF1 mouse embryos transported by parcel-delivery service.</p