Role of Site-Specific Glycosylation in the I-Like Domain of Integrin β1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation

Lin Cao,Zengqi Tan,Xiuxiu Wang,Feng Guan,Yurong Wu,Xiang Li

doi:10.3390/ijms22041770

Abstract

Integrin β1 plays an essential role in the crosstalk between tumor cells and their microenvironment. Aberrant N-glycosylation of integrin β1 was documented to alter integrin β1 expression, dimerization, and biological function. However, the biological function of site-specific N-glycosylation of integrin β1 on extracellular vesicles is not fully understood. In this study, we mutated putative N-glycosylation sites in different domains of integrin β1. Removal of the N-glycosylation sites on the I-like domain of integrin β1 (termed the Δ4–6 β1 mutant) suppressed focal adhesion kinase (FAK) signaling, cell migration, and adhesion compared with other β1 mutants. Cell adhesion, migration, and activation of FAK were suppressed in recipient MCF7 cells co-cultured with Δ4–6 mutant cells and treated with small extracellular vesicles (sEVs) from Δ4–6 mutant cells. Notably, the wild-type and β1 mutant were both present in sEVs, and could be transferred to recipient cells via sEVs, resulting in changes of cell behavior. Our findings demonstrate the important roles of N-glycosylation of the I-like domain of integrin β1. Moreover, the vesicular Δ4–6 β1 mutant can regulate integrin-mediated functions in recipient cells via sEVs.

Highlights

Small extracellular vesicles, one type of membrane-covered structure, originate from the endosomal pathway, are secreted by exocytosis into the surrounding extracellular space, and are present in various body fluids [1,2]. small extracellular vesicles (sEVs) carry plentiful bioactive materials, including proteins, nuclear acids, and lipids, which mediate various types of cell-to-cell communication [3]
Vesicular integrins determine the organ-specific metastasis of derived tumor cells, and blockage of integrin binding with extracellular matrix (ECM) was found to decrease sEV uptake and tumor metastasis [7]
Previous study had illustrated that 12 N-glycosylation sites characterized by asparagine residues within the NX(S/T) consensus sequence exist on integrin β1

Summary

Introduction

Small extracellular vesicles (sEVs), one type of membrane-covered structure (diameter30–100 nm), originate from the endosomal pathway, are secreted by exocytosis into the surrounding extracellular space, and are present in various body fluids [1,2]. sEVs carry plentiful bioactive materials, including proteins, nuclear acids, and lipids, which mediate various types of cell-to-cell communication [3]. Vesicular integrins determine the organ-specific metastasis of derived tumor cells, and blockage of integrin binding with extracellular matrix (ECM) was found to decrease sEV uptake and tumor metastasis [7]. Recent studies have documented that aberrant expression of integrin β1 and dysregulation of focal adhesion kinase (FAK)/protein kinase B (AKT) signaling closely correlate with the malignant phenotype [9,10]. Cell migration and invasion were enhanced by gamma-synuclein through the activation of the integrin β1–FAK signal pathway in colorectal cancer cells [11]. Aberrant glycosylation of integrin β1 affected its biological function and altered cell adhesion, migration, and survival in cancer [12]. Hou et al revealed that membrane-proximal N-glycosylation on integrin β1 could regulate cell migration by promoting integrin β1 activation [14]. Knockdown of O-GlcNAc transferase increased the focal adhesion complex formation of integrin β1 and FAK, as well as levels of active integrin β1, resulting in the promotion of cell adhesion and suppression of cell migration [15]

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Results

Discussion

Conclusion