N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha3beta1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta1,6-GlcNAc-branched N-glycans on integrin alpha3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.     

All-Trans-Retinoic Acid Modulates ICAM-1 N-Glycan Composition by Influencing GnT-III Levels and Inhibits Cell Adhesion and Trans-Endothelial Migration

Changes in the expression of glycosyltransferases directly influence the oligosaccharide structures and conformations of cell surface glycoproteins and consequently cellular phenotype transitions and biological behaviors. In the present study, we show that all-trans-retinoic acid (ATRA) modulates the N-glycan composition of intercellular adhesion molecule-1 (ICAM-1) by manipulating the expression of two N-acetylglucosaminyltransferases, GnT-III and GnT-V, via the ERK signaling pathway. Exposure of various cells to ATRA caused a remarkable gel mobility down-shift of ICAM-1. Treatment with PNGase F confirmed that the reduction of the ICAM-1 molecular mass is attributed to the decreased complexity of N-glycans. We noticed that the expression of the mRNA encoding GnT-III, which stops branching, was significantly enhanced following ATRA exposure. In contrast, the level of the mRNA encoding GnT-V, which promotes branching, was reduced following ATRA exposure. Silencing of GnT-III prevented the molecular mass shift of ICAM-1. Moreover, ATRA induction greatly inhibited the adhesion of SW480 and U937 cells to the HUVEC monolayer, whereas knock-down of GnT-III expression effectively restored cell adhesion function. Treatment with ATRA also dramatically reduced the trans-endothelial migration of U937 cells. These data indicate that the alteration of ICAM-1 N-glycan composition by ATRA-induced GnT-III activities hindered cell adhesion and cell migration functions simultaneously, pinpointing a unique regulatory role of specific glycosyltransferases in the biological behaviors of tumor cells and a novel function of ATRA in the modulation of ICAM-1 N-glycan composition.     

Modulation of ionizing radiation-induced apoptosis and cell cycle arrest by all-trans retinoic acid in promyelocytic leukemia cells (HL-60)

Acute promyelocytic leukemia is characterized by a block of myeloid differentiation. The incubation of cells with 1 micromol/l all-trans retinoic acid (ATRA) for 72 h induced differentiation of HL-60 cells and increased the number of CD11b positive cells. Morphological and functional changes were accompanied by a loss of proliferative capacity. Differentiation caused by preincubation of leukemic cells HL-60 with ATRA is accompanied by loss of clonogenicity (control cells: 870 colonies/10(3) cells, cells preincubated with ATRA: 150 colonies/10(3) cells). D0 for undifferentiated cells was 2.35 Gy, for ATRA differentiated cells 2.46 Gy. Statistical comparison of clonogenity curves indicated that in the whole range 0.5-10 Gy the curves are not significantly different, however, in the range 0.5-3 Gy ATRA differentiated cells were significantly more radioresistant than non-differentiated cells. When HL-60 cells preincubated with 1 micromol/l ATRA were irradiated by a sublethal dose of 6 Gy, more marked increase of apoptotic cells number was observed 24 h after irradiation and the surviving cells were mainly in the G1 phase of the cell cycle, while only irradiated cells were accumulated in G(2) phase. Our results imply that preincubation of cells with ATRA accelerates apoptosis occurrence (24 h) after irradiation by high sublethal dose of 6 Gy. Forty-eight hours after 6 Gy irradiation, late apoptotic cells were found in the group of ATRA pretreated cells, as determined by APO2.7 positivity. This test showed an increased effect (considering cell death induction) in comparison to ATRA or irradiation itself.     

Expression of ��1,3-N-acetylglucosaminyltransferases during differentiation of human acute myeloid leukemia cells

The expressions of β1,3-N-acetylglucosamonyltransferase-2 and -8 (β3GnT-2, β3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and β3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. β3GnT-2 and β3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while β3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of β3GnT-8 was more than β3GnT-2, while in NB4 cells treated with DMSO, the increase of β3GnT-2 was more than β3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of β3GnT-2 and β3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of β3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of β3GnT-2 and -8. The expression of β3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of β3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ     

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.     

Increased susceptibility to apoptosis of human hepatocarcinoma cells transfected with antisense N-acetylglucosaminyltransferase V cDNA.

The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2. 4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and beta-1,6-GlcNAc branching in the cell glycoproteins compared with the cells mock-transfected with the vector pcDNA3 (pcDNA3/7721). The growth rate of GnT-V-AS/7721 was decreased in serum-containing medium, while the cell death was accelerated in serum-free medium. The GnT-V-AS/7721 cells were more susceptible to the apoptosis induced by ATRA than the mock-transfected cells. This was evidenced by the obvious appearance of a hypoploid sub-G(1) fraction in the DNA histogram using FCM analysis, the more condensed new moon-type nuclei under morphological observation, and the more intensive TUNEL reaction for assaying the fragmented DNA. At the same time as GnT-V down-regulation by GnT-V-AS, an increase of another N-aceylglusaminyltransferase, GnT-III (EC 2.4.1.144), was observed, and the biological significance of this finding was discussed.     

Alteration in N -acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma

The activities of three N -acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E(4)-PHA. The increased GnT-V activity led to the elevation of the beta1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients.     

Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator

Glycosylation is one of the most abundant posttranslational modification reactions, and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharide structure (glycan) are associated with many physiological and pathological events, including cell adhesion, migration, cell growth, cell differentiation and tumor invasion. Glycosylation reactions are catalyzed by the action of glycosyltransferases, which add sugar chains to various complex carbohydrates such as glycoproteins, glycolipids and proteoglycans. Functional glycomics, which uses sugar remodeling by glycosyltransferases, is a promising tool for the characterization of glycan functions. Here, we will focus on the positive and negative regulation of biological functions of integrins by the remodeling of N-glycans with N-acetylglucosaminyltransferase III (GnT-III) and N-acetylglucosaminyltransferase V (GnT-V), which catalyze branched N-glycan formations, bisecting GlcNAc and β1,6 GlcNAc, respectively. Typically, integrins are modified by GnT-III, which inhibits cell migration and cancer metastasis. In contrast, integrins modified by GnT-V promote cell migration and cancer invasion.     

Regulation of progranulin expression in myeloid cells

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.     

Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNAc-transferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death

Human glioma cell line U-373 MG expresses CMP-NeuAc : Galbeta1,3GlcNAc alpha2,3-sialyltransferase [EC No. 2.4.99.6] (alpha2,3ST), UDP-GlcNAc : beta-d-mannoside beta1,6-N-acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT-V) and UDP-GlcNAc3: beta-d-mannoside beta1,4-N-acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT-III) but not CMP-NeuAc : Galbeta1,4GlcNAc alpha2,6-sialyltransferase [EC 2.4.99.1] (alpha2,6ST) under normal culture conditions. We have previously shown that transfection of the alpha2,6ST gene into U-373 cells replaced alpha2,3-linked sialic acids with alpha2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U-373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U-373 cell viability showed a three-fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2-ceramide or etoposide, when either alpha2,6ST and GnT-V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as alpha2,3ST and GnT-III, were able to decrease viability two-fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase-transfected U-373 cells to pro-apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase-3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U-373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3-kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.     

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.     

Cell-cell interaction-dependent regulation of N-acetylglucosaminyltransferase III and the bisected N-glycans in GE11 epithelial cells. Involvement of E-cadherin-mediated cell adhesion

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and alpha1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.     

Effects of dibutyryl cAMP and bromodeoxyuridine on expression ofN-acetylglucosaminyltransferases III and V in GOTO neuroblastoma cells

The sugar chain structures of the cell surface change dramatically during cellular differentiation. A human neuroblastoma cell line, GOTO, is known to differentiate into neuronal cells and Schwannian cell-like cells on treatments with dibutyryl cAMP and bromodeoxyuridine, respectively. We have examined the expression of UDP-N-acetylglucosamine: -d-mannoside -1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) and UDP-N-acetylglucosamine: -6-d-mannoside -1,6N-acetylglucosaminyltransferase V (GnT-V: EC 2.4.1.155), two major branch forming enzymes inN-glycan synthesis, in GOTO cells on two distinct directions of differentiation.In neuronal cell differentiation, GnT-III activity showed a slight increase during initial treatment with Bt₂cAMP for 4 days and decreased drastically after the fourth day, but the mRNA level of GnT-III did not show a decrease but in fact a slight increase. GnT-V activity increased to approximately two- to three-fold the initial level with increasing mRNA level after 8 days, and lectin blot analysis showed an increase in reactivity toDatsura stramonium (DSA) of the immunoprecipitated neural cell adhesion molecule (NCAM). In Schwannian cell differentiation, the activity and mRNA level of GnT-III showed no significant change on treatment with BrdU. GnT-V activity also showed no change in spite of the gradual increase in the mRNA level. These results suggest that the activation of GnT-V during neuronal cell differentiation of GOTO cells might be a specific change for branch formation in N-glycans, and this affects the sugar chain structures of some glycoproteins such as NCAM.Abbreviations and trivial names GnT N-acetylglucosaminyltransferase - Bt₂cAMP N ⁶,O ⁶-dibutyryl cAMP - BrdU bromodeoxyuridine - DSA Datsura stramonium - NCAM neural cell adhesion molecule - PAGE polyacrylamide gel electrophoresis     

Changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (HL-60) during monocytoid differentiation and myeloid differentiation. Appearance of high mannose-type oligosaccharides in neutral fraction

Structural changes in the asparagine-linked sugar chains of plasma membrane glycoproteins during myeloid and monocytoid differentiation were investigated by the use of an in vitro differentiation system for human promyelocytic leukemic cells (HL-60), which can be induced to more mature myeloid cells by exposure to dimethyl sulfoxide and to macrophage-like cells by a phorbol ester. The asparagine-linked sugar chains released from their plasma membranes by hydrazinolysis were separated into a neutral fraction and an acidic fraction composed of over ten components. The content of neutral oligosaccharides, which accounted for 8% of the total asparagine-linked sugar chains in HL-60 cells, increased slightly to 13% in dimethyl sulfoxide-induced cells and markedly to 33% in phorbol ester-induced cells. Structural analyses revealed that the neutral oligosaccharides of HL-60 cells are all of the complex type with a variety of Gal beta 1----4GlcNAc beta 1----units in their outer chain moieties and the following core structure: (sequence; see text) After myeloid and monocytoid differentiation, the total amount of neutral complex-type sugar chains did not change significantly, but newly found high mannose-type sugar chains contributed up to 3% and 24% of the total sugar chains, respectively.     

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.