Language：English   Publisher：Biochemical and Biophysical Research Communications  

Sialic acids (Sias) are often linked to galactose (Gal) residues by α2,6- and α2,3-linkages in glycans of glycoproteins. Sias are indispensable for vertebrate development, because organisms deficient in some enzymes in the Sia synthetic pathway are lethal during the development. However, it remains unknown if the difference of Siaα2,6Gal or α2,3Gal linkage has a critical meaning. To find a clue to understand significance of the linkage difference at the organism level, medaka was used as a vertebrate model. In embryos, Siaα2,6Gal epitopes recognized by Sambucus nigra lectin (SNA) and Siaα2,3Gal epitopes recognized by Maackia amurensis lectin (MAA) were enriched in the blastodisc and the yolk sphere, respectively. When these lectins were injected in the perivitelline space, SNA, but not MAA, impaired embryo body formation at 1 day post-fertilization (dpf). Most Siaα2,6Gal epitopes occurred on N-glycans owing to their sensitivity to peptide:N-glycanase. Of knockout-medaka (KO) for either of two β-galactoside:α2,6-sialyltransferase genes, ST6Gal I and ST6Gal II, only ST6Gal I–KO showed severe cardiac abnormalities at 7–16 dpf, leading to lethality at 14–18 dpf. Interestingly, however, these cardiac abnormalities of ST6Gal I–KO were rescued not only by forced expression of ST6Gal I, but also by that of ST6Gal II and the β-galactoside:α2,3-sialyltransferase IV gene (ST3Gal IV). Taken together, the Siaα2,6Gal linkage synthesized by ST6Gal I are critical in heart development; however, it can be replaced by the linkages synthesized by ST6Gal II and ST3Gal IV. These data suggest that sialylation itself is more important than its particular linkage for the heart development.

DOI： 10.1016/j.bbrc.2023.01.010

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Language：English   Publisher：Biomolecules  

Alveolin is a cortical alveolus proteinase that is secreted in the perivitelline space (PVS) at fertilization to act on the chorion. Purified alveolin is known to induce chorion hardening in vitro by processing zona pellucida B (ZPB), a major chorion component. However, in vivo function of alveolin remains unclear; thus, in this study, the effects of alveolin efficiency (Alv−/−) at the organism level were investigated using the medaka, Oryzias latipes. The Alv−/− fertilized eggs were mechanically fragile; however, they developed normally and left offspring as long as they were carefully handled before hatching. A mechanical press test showed that the Alv−/− fertilized eggs were six times more fragile than the wild-type eggs. They were 35% larger owing to the enlarged PVS, 34% thinner, and permeable to even 10 kDa FITC-dextran. These results are consistent with the transmission electron microscopy observation that the periphery of the inner layers was highly porous in the Alv−/− chorion. In chorion hardening, the alveolin-mediated processing of ZPB and the transglutaminase (TGase)-mediated crosslinking of chorion components are the key steps. This study was the first to show that alveolin also processed TGase concomitantly with ZPB, which greatly facilitated the crosslinking. Thus, alveolin was concluded to be the primary trigger for chorion hardening in vivo. Furthermore, fertilization in a balanced salt solution could partially improve the impaired chorion hardening of the Alv−/− eggs fertilized in water, probably through an alveolin-independent mechanism.

DOI： 10.3390/biom13010146

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Language：English   Publisher：Glycobiology  

Sialic acid (Sia) is a group of acidic sugars with a 9-carbon backbone, and classified into 3 species based on the substituent group at C5 position: N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). In Escherichia coli, the sialate aldolase or N-acetylneuraminate aldolase (NanA) is known to catabolize these Sia species into pyruvate and the corresponding 6-carbon mannose derivatives. However, in bacteria, very little is known about the catabolism of Kdn, compared with Neu5Ac. In this study, we found a novel Kdn-specific aldolase (Kdn-aldolase), which can exclusively degrade Kdn, but not Neu5Ac or Neu5Gc, from Sphingobacterium sp., which was previously isolated from a Kdn-assimilating bacterium. Kdn-aldolase had the optimal pH and temperature at 7.0-8.0 and 50 °C, respectively. It also had the synthetic activity of Kdn from pyruvate and mannose. Site-specific mutagenesis revealed that N50 residue was important for the Kdn-specific reaction. Existence of the Kdn-aldolase suggests that Kdn-specific metabolism may play a specialized role in some bacteria.

DOI： 10.1093/glycob/cwac053

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Language：English   Publisher：Biochemical and Biophysical Research Communications  

The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by 31P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

DOI： 10.1016/j.bbrc.2022.05.031

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Language：English   Publisher：Scientific Reports  

Glycosylation of proteins and lipids occurs in vertebrates, usually terminating with sialylation, which regulates the physicochemical and biological properties of these glycoconjugates. Although less commonly known, sialic acid residues also undergo various modifications, such as acetylation, methylation, and sulfation. However, except for acetylation, the enzymes or functions of the other modification processes are unknown. To the best of our knowledge, this study is the first to demonstrate the ubiquitous occurrence of sulfated sialic acids and two genes encoding the sialate: O-sulfotransferases 1 and 2 in vertebrates. These two enzymes showed about 50% amino acid sequence identity, and appeared to be complementary to each other in acceptor substrate preferences. Gene targeting experiments showed that the deficiency of these genes was lethal for medaka fish during young fry development and accompanied by different phenotypes. Thus, the sulfation of sialic acids is essential for the vertebrate development.

DOI： 10.1038/s41598-022-15143-4

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