Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization

Abstract

BackgroundInsufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer's disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.ResultGenetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.ConclusionThe novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer’s disease and atherosclerosis.

A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function

“…5 Others have reported similar findings when expressing artificial constructs of SRA. 19 We have demonstrated that MARCO and MARCOII are expressed at comparable levels in our transient transfection system by western blotting and that they both expressed on the cell surface by flow cytometry (Figures 2a and b) and IF microscopy (Figures 2c-j). Therefore, differences in function are not due to differences in protein expression.…”

“…Previous work by Brännström et al 5 highlighted the importance of the RxR motif within the SRCR domain of MARCO for ligand binding using artificially truncated or mutated constructs; however, some constructs were not expressed on the cell surface 5 . Others have reported similar findings when expressing artificial constructs of SRA 19 . We have demonstrated that MARCO and MARCOII are expressed at comparable levels in our transient transfection system by western blotting and that they both expressed on the cell surface by flow cytometry (Figures 2a and b) and IF microscopy (Figures 2c and j).…”

Understanding molecular interactions between scavenger receptor A and its natural product inhibitors through molecular modeling studies

“…18 Recently the cysteine rich domain of SRA was shown to also serve as a potential binding site for Ac-LDL. 20 We recently completed the construction of SRA cysteine rich domain homology models 18 based on the available crystal structures of the cysteine rich domain of MARCO, another member of class A scavenger receptor family, in its monomeric and dimeric form (PDB: 2OY3 and 2OYA). 21 Mouse scavenger receptor MARCO had the highest sequence identity (43%) and homology (63%) to SRA and the fact that both proteins belong to the same receptor family, made the crystal structure of MARCO a suitable choice as the template for homology modeling.…”

Identifying N-linked glycan moiety and motifs in the cysteine-rich domain critical for N-glycosylation and intracellular trafficking of SR-AI and MARCO

“…Activated scavenger receptor A (SR-A) promotes glial internalization of Amyloid-β peptide (Aβ), which is a key histopathological characteristic of Alzheimer’s disease (AD) [ 1 , 2 ]. Previously, we identified SR-A as the prominent subtype of scavenger receptor mediating oligomeric Aβ (oAβ) internalization in microglia, and revealed that the cysteine-rich (SRCR) domain of SR-A type I (SR-AI) may be the critical domain on modulating the efficacy of surface targeting and mediating oAβ internalization [ 3 , 4 ].…”