Catalytic activity of Setd2 is essential for embryonic development in mice: establishment of a mouse model harboring patient-derived Setd2 mutation.

SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2CD/CD and Setd2-/- mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2-/-, the Setd2CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5' Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions.

N-glycosylation at N57/100/110 affects CD44s localization, function and stability in hepatocellular carcinoma.

The glycosylation levels of proteins in cancer cells are closely related to cancer invasion and migration. CD44 is a transmembrane glycoprotein that is significantly overexpressed in a variety of tumor cells and has been proven to promote the migration and motility of cancer cells, but the effect of its N-glycosylation modification on CD44 protein function in tumors is less studied. Here, we investigated the effect of six N-glycan chains (N25/57/100/110/120/255) on CD44s localization, function and stability in hepatocarcinoma cells. When the six sites were mutated, we found that CD44s lost its membrane localization in Huh7 and MHCC-97H cells. On this basis, we identified three glycosylation sites on CD44s (N57, N100 and N110) that played key roles in intracellular localization. When N57, N100 and N110 were mutated together, CD44 localized to the cytoplasm, while another three-site mutant (N25/N120/N255) was still anchored to the membrane. In addition, the ability of CD44-N57Q/N100Q/N110Q to promote the metastasis and invasion of Huh7 and 97H cells was weakened compared with that of CD44-N25Q/N120Q/N255Q. Furthermore, CD44-N57Q/N100Q/N110Q accumulated abnormally in the ER, and a high level of the ER stress (ERS) marker BiP was detected at the same time compared with wild-type CD44. When the lysosome inhibitor CQ was added, the content of mutant protein that triggered ERS significantly increased, which indicated that the degradation mode of CD44-N57Q/N100Q/N110Q after ERS was mainly through the lysosomal pathway (ERLAD). The results revealed that the N-glycosylation sites N57, N100 and N110 mutated on CD44s affected its function and degraded it by lysosomes after triggering ERS. These findings provide data for new studies on ER-related degradation, further promote the study of the glycan chain function of CD44 and furnish new ideas for the treatment of liver cancer metastasis.

NEU4 inhibits motility of HCC cells by cleaving sialic acids on CD44.

Hepatocellular carcinoma (HCC) is an extremely metastatic tumor. Sialic acids (SAs) are associated with cancer development and metastasis. NEU4 is a sialidase that removes SAs from glycoconjugates, while the function of the NEU4 in HCC has not been clearly explored. In our research, we found the NEU4 expression was significantly down-regulated in HCC tissues, which was correlated with high grades and poor outcomes of HCC. The NEU4 expression could be regulated by histone acetylation. In the functional analysis of NEU4, the cell motility was inhibited when NEU4 was overexpressed, and restored when NEU4 expression was down-regulated. Similarly, NEU4 over-expressed HCC cells showed less metastasis in athymic nude mice. Further study revealed that NEU4 could inhibit cell migration by enzymatic decomposition of SAs. Our results verified a NEU4 active site (NEU4E235) and overexpressing inactivates NEU4E235A that weakens the inhibition ability to cell migration. Further, 70 kinds of specific interacting proteins of NEU4 including CD44 were identified through mass spectrum. Moreover, the α2,3-linked SAs on CD44 were decreased and the hyaluronic acid (HA) binding ability was increased when NEU4 over-expressed or activated. Additionally, the mutation of CD44 with six N-glycosylation sites showed less sensibility to NEU4 on cell migration compared with wild-type CD44. In summary, our results revealed the mechanism of low expression of NEU4 in HCC and its inhibitory effect on cell migration by removal of SAs on CD44, which may provide new treatment strategies to control the motility and metastasis of HCC.

A sweet spot for macrophages: Focusing on polarization.

Macrophages are a type of functionally plastic cells that can create a pro-/anti-inflammatory microenvironment for organs by producing different kinds of cytokines, chemokines, and growth factors to regulate immunity and inflammatory responses. In addition, they can also be induced to adopt different phenotypes in response to extracellular and intracellular signals, a process defined as M1/M2 polarization. Growing evidence indicates that glycobiology is closely associated with this polarization process. In this research, we review studies of the roles of glycosylation, glucose metabolism, and key lectins in the regulation of macrophages function and polarization to provide a new perspective for immunotherapies for multiple diseases.