Abstract
The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.
Highlights
Control the structure and metabolism of high density lipoprotein (HDL) by mediating the transport of lipids from HDL to cells
We have examined the consequences of mutating each of these sites (Asn 3 Gln or Thr 3 Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities
Identification of N-Linked Glycosylation Sites on mSR-BI by Mutagenesis—Murine SR-BI contains 11 potential N-linked glycosylation sites (Asn-X-Ser/Thr) in its extracellular domain [1]; these include the asparagines at positions: 102, 108, 116, 173, 212, 227, 255, 288, 310, 330, and 383
Summary
Materials—[1␣,2␣(n)-3H]Cholesteryl oleoyl ether ([3H]CEt, 1 mCi/ ml, specific activity of 58 Ci/mmol) was from Amersham Biosciences. On day 1, cells were transfected with the plasmid expression vectors for wild-type or mutant mSR-BI, or with a control plasmid that did not encode a protein product (pCDNA1), using the DEAE-dextran method described previously [31]. For two-color flow cytometry, cells were first labeled with DiI-HDL at 37 °C as described above, washed twice with PBS, and incubated with either Alexa-HDL or anti-mSR-BI KKB-1 antiserum and FITCconjugated goat anti-rabbit IgG as described above. Receptor-specific values for DiI uptake, Alexa-HDL binding, and surface expression are defined as the differences between the values determined using cells transfected with the plasmids encoding the wild-type or mutant receptors and those determined using cells transfected with the non-coding (empty vector) controls. Images were saved as tiff format files and edited with Photoshop 6.0 software
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