Study On The Mechanism Of The Proliferation Of Myeloid Maligant Clone Cells Affected By Bisecting GlcNAc In Bone Marrow Stromal Cells

Hematopoietic stem cells can produce all lineages of blood cells,which are mainly present in the bone marrow,and bone marrow niche can provide the regulatory signals that are necessary for the quiescent,proliferation,and differentiation of hematopoietic stem cells(HSCs).Bone marrow stromal cells are the main component of the bone marrow,which can not only act as "silent companions" of hematopoietic stem cells and secrete a variety of positive and negative regulatory factors required for hematopoietic processes,but also participate in the forming of favorable space for HSCs to mature.Melanoma cell adhesion molecule(MCAM)is considered to be a marker of bone marrow niche and a decision maker for differentiation of bone marrow stromal cells.Glycosylation is one of the most common post-translational modifications of proteins,which is closely related to expression,localization and half-life of many proteins,and can influence cell proliferation or apoptosis.Carbohydrate chains are produced by the combined action of glycosyltransferases and glycosidases.Among them,N-acetylglucosamine aminotransferase III(MGAT3)can catalyze the form of bisecting GlcNAc by linking the ?-1,4 glycosidic linkage of GlcNAc to the ?-mannose of the N-glycan.Bisecting GlcNAc can inhibit the adhesion between cells and extracellular matrix to inhibit tumor metastasis,but its effect on the bone marrow microenvironment and cloned cells in the bone marrow microenvironment has not been thoroughly studied.The co-culture model of bone marrow stromal cell lines with clonal cells is often used to study the interaction between stromal cells and myeloid malignant clone cells in the bone marrow microenvironment.Previous data has shown that the expression of MGAT3 and MCAM is significantly different in stromal cells HS27 a and HS5.In HS27 a cells,the expression of MGAT3 is low but the expression of MCAM is very high.On the contrary,in HS5 cells,the expression of MGAT3 is high but MCAM is hardly expressed.The published data has shown that in co-culture models,overexpression of MCAM in HS5 cells can promote the proliferation of malignant cloned cells,while inhibition of MCAM in HS27 a cells can inhibit the proliferation of malignant cloned cells.Our clinical data showed that expression of MGAT3 is lower in patients with MDS than in controls.Meanwhile,co-culture with malignant cloned cells inhibited the expression of bisecting GlcNAc in bone marrow stromal cells.We speculate that bisecting GlcNAc plays an important role in the bone marrow microenvironment.Then the function and mechanism of bisecting GlcNAc in bone marrow microenvironment is studied.We found that overexpression of MGAT3(HS27a-MGAT3)in HS27 a cells can inhibit the proliferation of co-cultured malignant cloned cells,while inhibition of MGAT3(HS5-sh MGAT3)in HS5 cells can promote the proliferation of co-cultured malignant cloned cells.We further found that over-expressing MGAT3 in HS27 a cells results in downregulated expression of MCAM,and inhibition of MGAT3 in HS5 cells can lead to upregulated expression of MCAM.Next,we want to prove the relationship of MGAT3 and MCAM in conditional expressed MGAT3 cells and primary cells.We construct doxycyclineregulated MGAT3 expression in HS27 a cells(HS27a-Tet One-MGAT3),and successfully cultured mouse bone marrow mesenchymal stem cells(MSC)in vitro and then constructed MSC cells that over-expressing MGAT3(MSC-MGAT3)or inhibiting MGAT3(MSCsh MGAT3).It was found that adding doxycycline resulted in the gradually overexpressed MGAT3 with gradually down-regulated expression of MCAM in HS27a-Tet One-MGAT3.Moreover,expression of MCAM is down-regulated in MSC-MGAT3 but up-regulated in MSC-sh MGAT3.All of these results indicate that the expression of MGAT3 and MCAM is reversed.Co-immunoprecipitation experiments and mass spectrometry results showed the direct modification of the bisecting GlcNAc on MCAM,and the modified site occurred on the N of peptide sequence QGNLS.The biotin-labeled membrane protein experiments showed that after overexpressed MGAT3 in HS27 a cells,the total MCAM protein was downregulated,but the expression of MCAM was up-regulated in the cytoplasma,and it was down-regulated on the surface of cellular membrane.Moreover,MCAM colocalizes with Golgi and lysosomes in HS27a-MGAT3 cells,which indicates that overexpression of MGAT3 in cells may prevent MCAM from becoming a mature membrane protein and flipping out of the cellular membrane,then leading to the degradation of MCAM in lysosomes.Finally,in mouse model,we found that HS27a-MGAT3 cells can inhibit the proliferation of KG1 a in spleen,and the survival rate of this group is prolonged.Overall,our data showed that MGAT3 and its synthetized bisecting GlcNAc play an important role in the bone marrow microenvironment.MGAT3 may further affect the proliferation of myeloid malignant clonal cells by affecting the expression of the bone marrow niche marker MCAM.