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

Abstract

Background Information: Like most other cell surface proteins, α 5 β 1 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 α 5 β 1 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 β 1 integrin. The extensive glycan information that results from our study was projected onto a map of α 5 β 1 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 α 5 β 1 integrin. A striking example concerns the involvement of glycanbinding 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 α 5 β 1 integrin activity and endocytic trafficking. Significance: Glycosylation of α 5 β 1 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 α 5 β 1 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.

Spatial N-glycan rearrangement on α5β1integrin nucleates galectin-3 oligomers to determine endocytic fate

“…The complex glycan at a5 chain position N356 (Mirgorodskaya et al, 2022) of the headpiece (N307 in human, Figure S8E) also points towards the additional cryo-EM density that we ascribed to Gal3 (Figure 6E). Based on GlycoSHIELD modeling, it was indeed possible to position another Gal3 CRD for interaction with a galactose residue on the N356 glycan, resulting in a Gal3 dimer on the N356 and N918 glycan that forms a bridge between headpiece and leg piece (Figure 6H, right).…”

“…Cross-linking mass spectrometry revealed the proximity between position 227 in the CRD of Gal3 with positions in the membrane distal and membrane-proximal calf regions of the leg piece of α 5 integrin (Figures 6F and S8A). Several N-glycans at sites close to cross-linking positions, i.e., N642, N761, N773, N822, and N917/N918 of the α 5 chain, are complex-type multi-antennae structures that are of galectin-binding competent nature, as determined by site-specific glycoproteomics on the same α 5 β 1 integrin preparation from rat liver as used in the current study (Mirgorodskaya et al, 2022). Using GlycoSHIELD (Gecht et al, BioRxiv), possible conformations of these N-glycans were projected onto the α 5 β 1 integrin model (Figures 6G and S8B).…”

“…To structurally characterize Gal3 binding to α 5 β 1 integrin via cryogenic electron microscopy (cryo- EM), we first vitrified peptidisc-embedded α 5 β 1 integrin alone (Figure S5A) and refined the structure of the inactive bent-closed conformation to resolutions of 3.7 Å in the headpiece and 4.7 Å in the leg piece, respectively (Figures S5B-J). A homology model of rat α 5 β 1 integrin (Mirgorodskaya et al, 2022) was fitted individually into the density maps of the headpiece (flexible fit, Figure 6D and Figures S5K and S5L) and leg piece (rigid body fit, Figure 6D). The structure revealed a bent-closed conformation with an angle between headpiece and leg piece that is larger than for the bent-closed conformations of other integrins (Adair et al, 2023), and similar to human α 5 β 1 integrin (Schumacher et al, 2021).…”

“…A previously generated homology model of the extracellular domain of rat α 5 β 1 integrin (residues 94 - 1041 and 23 - 719, respectively) (Mirgorodskaya et al, 2022) based on human α 5 β 1 integrin (PDB 7NXD) (Schumacher et al, 2021) was used as starting model. The homology model was split into headpiece and leg piece (α 5 , residues 94 - 691 and 692 - 1041; β 1 , residues 23 - 504 and 505 - 720, respectively) and rigid-body fitted into the sharpened density maps of headpiece and leg piece using UCSF Chimera (Pettersen et al, 2004), followed by flexible fitting with imodfit (Lopez-Blanco and Chacon, 2013).…”

Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck

“…Also, the presence of O-acetyl Neu5Ac was not detected. This hydrolysis-sensitive modification may be lost during the mild alkaline conditions used, although it is unlikely in this case since the Sia structure was abundantly detected in another study using similar preparation conditions ( Mirgorodskaya et al. 2022 ).…”