Site-specific N-glycan profiles of α5β1 integrin from rat liver

Abstract

Background Information

Like most other cell surface proteins, α₅β₁ integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined.

Results

Here, we have established the first comprehensive site-specific glycan map of α₅β₁ integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of β₁ integrin. The extensive glycan information that results from our study was projected onto a map of α₅β₁ integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α₅β₁ integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis.

Conclusion

We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α₅β₁ integrin activity and endocytic trafficking.

Significance

Glycosylation of α₅β₁ integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α₅β₁ integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of “native” glycoproteins.