Yoshinobu Kariya

N-Acetylglucosaminyltransferase III Antagonizes the Effect of N-Acetylglucosaminyltransferase V on α3β1 Integrin-mediated Cell Migration

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of β1,6-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 α3β1 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 β1,6-GlcNAc-branched N-glycans on integrin α3 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 β1,6-GlcNAc-branched N-glycans on the α3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the α3 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.

Deletion of Core Fucosylation on α3β1 Integrin Down-regulates Its Functions

The core fucosylation (α1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently α1,6-fucosylation has been further reported to be very crucial by the study of α1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on α3β1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in α3β1 integrin-mediated events has been studied by using Fut8+/+ and Fut8–/– embryonic fibroblasts, respectively. We found that the core fucosylation of α3β1 integrin, the major receptor for laminin 5, was abundant in Fut8+/+ cells but was totally abolished in Fut8–/– cells, which was associated with the deficient migration mediated by α3β1 integrin in Fut8–/– cells. Moreover integrin-mediated cell signaling was reduced in Fut8–/– cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of α3β1 integrin.

Potential roles of N-glycosylation in cell adhesion

The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins; the adhesion receptors, the cell-extracellular matrix (ECM) proteins, and the cytoplasmic plaque/peripheral membrane proteins. The cell adhesion receptors are usually transmembrane glycoproteins (for example E-cadherin and integrin) that mediate binding at the extracellular surface and determine the specificity of cell-cell and cell-ECM recognition. E-cadherin-mediated cell-cell adhesion can be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. On the other hand, integrin-mediated cell-ECM interactions play important roles in cytoskeleton organization and in the transduction of intracellular signals to regulate various processes such as proliferation, differentiation and cell migration. ECM proteins are typically large glycoproteins, including the collagens, fibronectins, laminins, and proteoglycans that assemble into fibrils or other complex macromolecular arrays. The most of these adhesive proteins are glycosylated. Here, we focus mainly on the modification of N-glycans of integrins and laminin-332, and a mutual regulation between cell adhesion and bisected N-glycan expression, to address the important roles of N-glycans in cell adhesion.

Regulation of proliferation and chondrogenic differentiation of human mesenchymal stem cells by laminin-5 (Laminin-332)

Laminin‐5 (laminin‐332) is an important basement membrane protein that regulates cell attachment and motility. Recent studies have shown that laminin‐5 is expressed in human mesenchymal stem cells (MSCs) in culture and that the laminin γ2 chain is transiently expressed in chondrocytes during development. These studies suggest that laminin‐5 may be involved in the regulation of chondrogenic differentiation of MSCs. In this study, we examined a possible role of laminin‐5 in the proliferation and differentiation of human MSCs. When MSCs were incubated in the presence of a coated or soluble form of laminin‐5 in a growth medium, they proliferated more rapidly than nontreated cells, keeping their differentiation potential. On the other hand, laminin‐5 potently suppressed the chondrogenic differentiation of MSCs. These activities were mediated mainly by integrin α3β1. However, laminin‐5 had no effect on the osteogenic differentiation of MSCs. These results suggest that laminin‐5 may contribute to the development of bone tissues by promoting the proliferation and by suppressing the chondrogenic differentiation of MSCs.

A Mutual Regulation between Cell−Cell Adhesion and N-Glycosylation: Implication of the Bisecting GlcNAc for Biological Functions

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. E-cadherin-mediated cell-cell adhesion is believed to be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. Here, we focus mainly on a mutual regulation between E-cadherin-mediated cell-cell adhesion and N-acetylglucosaminyltransferase III (GnT-III) expression, and discuss its implications for biological functions.

Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility

Laminin-332 (Lm332; formerly laminin-5) is a basement membrane protein in the skin, which promotes cell motility in wound healing and cancer invasion. In a previous study, we reported that the introduction of bisecting GlcNAc into Lm332 (GnT-III-Lm332), catalyzed by N-acetylglucosaminyltransferase III (GnT-III), reduced cell migration (Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008) J. Biol. Chem. 283, 33036–33045). However, the underlying molecular mechanism by which GnT-III-Lm332 suppresses the normal biological functions of Lm332 remains to be elucidated. In this study, we show that galectin-3, which is a β-galactoside-binding protein, strongly bound to unmodified Lm332 but not to GnT-III-Lm332 and that binding of galectin-3 was completely blocked by lactose. Exogenous galectin-3 significantly enhanced keratinocyte cell motility on control Lm332 but not on GnT-III-Lm332. A functional blocking antibody against galectin-3 inhibited Lm332-induced α3β1 and α6β4 integrin clustering and focal contact formation. Co-immunoprecipitation revealed that galectin-3 associated with both β4 integrin and epidermal growth factor receptor, thereby cross-linking the two molecules. The associations were inhibited by either the presence of lactose or expression of GnT-III. Moreover, galectin-3 consistently enhanced ERK activation. Taken together, the results of this study are the first to clearly identify the molecular mechanism responsible for the inhibitory effects of GnT-III on extracellular matrix-integrin-meditated cell adhesion, migration, and signal transduction. The findings presented herein shed light on the importance of N-glycosylation-mediated supramolecular complex formation on the cell surface.

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of α3 chain

The basement membrane protein laminin‐5 promotes cell adhesion and migration. The carboxyl‐terminal G3 domain in the α3 chain is essential for the unique activity of laminin‐5. To investigate the function of the G3 domain, we prepared various recombinant laminin‐5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl‐terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ‐1 cells. This change was attributed to the loss of Lys‐Arg‐Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin‐5 receptors showed that integrins α3β1, α6β1, and α6β4 had different but synergistic effects on cell adhesion and migration on laminin‐5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin‐5 plays a central role to produce different biological effects on cells.

Laminin-6 Is Activated by Proteolytic Processing and Regulates Cellular Adhesion and Migration Differently from Laminin-5

Laminin-6 (LN6) and laminin-5 (LN5), which share the common integrin-binding domain in the laminin α3 chain, are thought to cooperatively regulate cellular functions, but the former has poorly been characterized. Human fibrosarcoma HT1080 cells expressing an exogenous α3 chain were found to secrete LN6 with the full-length α3 chain and a smaller amount of its processed form lacking the carboxyl-terminal G4-5 domain, besides mature LN5 without G4-5 (mat-LN5). We prepared the unprocessed LN6 and mat-LN5, as well as LN6 mutants without G4-5 (LN6ΔG4-5) or G5 (LN6ΔG5). These laminins supported attachment of HT1080 cells and human keratinocytes (HaCaT) through integrins α3β1 and/or α6β1. LN6ΔG4-5, LN6ΔG5, and mat-LN5 promoted rapid cell spreading, whereas LN6 did hardly. A purified G4-5 fragment of the laminin α3 chain supported cell attachment through interaction with heparan sulfate proteoglycans and promoted cell spreading in combination with mat-LN5 or LN6ΔG4-5. These results imply that the G4-5 domain within the LN6 molecule suppresses cell adhesion, while the released G4-5 promotes it. The presence of G5 rather than the heparin-binding domain G4 was responsible for the impaired cell spreading activity of LN6. However, the unprocessed LN6 promoted cell spreading in the presence of mat-LN5. Unlike mat-LN5, both LN6ΔG4-5 and LN6 did weakly or did not stimulate cell motility. These findings demonstrate that LN6 and LN5 have distinct biological activities, but they may cooperatively support cell adhesion. The proteolytic processing of the α3 chain seems to regulate the physiological functions of LN6.

Regulation of Biological Activity and Matrix Assembly of Laminin-5 by COOH-terminal, LG4–5 Domain of α3 Chain

The basement membrane protein laminin-5 (LN5; α3β3γ2) undergoes specific proteolytic processing of the 190-kDa α3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4–5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa α3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type α3 chain was completely cleaved, whereas the mutated α3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4–5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa α3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4–5 from the 190-kDa α3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4–5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4–5 synergistically enhances integrin signaling as it is released from the precursor LN5.

Regulation of biological activity of laminin‐5 by proteolytic processing of γ2 chain

Laminin‐5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino‐terminal region of the γ2 chain as well as at a carboxyl‐terminal region of the α3 chain. To clarify the biological effect of the γ2 chain processing, we prepared a human recombinant LN5 with the 150‐kDa, non‐processed γ2 chain (GAA‐LN5) and natural LN5 with the 105‐kDa, processed γ2 chain (Nat‐LN5). Comparison of their biological activities demonstrated that GAA‐LN5 had an about five‐times higher cell adhesion activity but an about two‐times lower cell migration activity than Nat‐LN5. This implies that the proteolytic processing of LN5 γ2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non‐processed γ2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed γ2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the γ2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.