Role of glycosylation and membrane environment in nicotinic acetylcholine receptor stability

Abstract

The effects of glycosylation and membrane environment on the structural stability of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated to improve our understanding of factors that influence eukaryotic membrane protein crystallization. Gel shift assays and carbohydrate-specific staining show that the deglycosylation enzyme, Endo F1, removes at least 50% of membrane-reconstituted nAChR glycosylation. The extent of deglycosylation with Endo F1 increases upon detergent solubilization. Removal of between 60-100% of high mannose moieties from the nAChR has no effect on nAChR secondary structure, stability, or flexibility. Deglycosylation does not influence either agonist binding or the ability of the nAChR to undergo agonist-induced conformational change. In contrast, nAChR structural stability, flexibility, and function are all negatively influenced by simple changes in reconstituted membrane lipid composition. Our results suggest that deglycosylation may represent a feasible approach for enhancing the crystallizability of the nAChR. Our data also demonstrate that the dependence of nAChR structural stability on lipid environment may represent a significant obstacle to nAChR crystallization. Some membrane proteins may have evolved complex interactions with their lipid environments. Understanding the complexity of these interactions may be essential for devising an appropriate strategy for the crystallization of some membrane proteins.

FIGURE 1

SDS-PAGE of decylmaltoside solubilized nAChR incubated with (A) sialidase, (B) PNGase F, (C) Endo F1, (D) sialidase and PNGase F, (E) sialidase and Endo F1, (F) PNGase F and Endo F1, and (G) sialidase, PNGase F and Endo F1 for 1, 4 and 8 days. For the individual enzyme experiments, the nAChR (20 mg/ml) was mixed 75:4 (v/v, nAChR/Endo F1 or sialidase) or 75:2 (v/v, nAChR/PNGase F) with each enzyme as supplied by the manufacturer. For the combination experiments, nAChR was mixed 75:2:2 (v/v/v, nAChR/glycosidase 1/glycosidase 2) or 75:2:2:2 (v/v/v/v, nAChR/glycosidase 1/glycosidase 2/glycosidase 3). The controls on the left of each panel have not been incubated with enzyme. The standards shown correspond to MWs of 116, 80, 51.8, and 34.7 kDa from top to bottom, respectively. The positions of the nAChR subunits are indicated on the left.

FIGURE 2

Deglycosylation of the nAChR reconstituted into soybean asolectin membranes. (A) SDS-PAGE of glycosylated (i) and Endo F1 deglycosylated (ii) nAChR. (B) SDS gel stained with a carbohydrate specific stain. The leftmost lane in (B) is a glycoprotein positive control (horse radish peroxidase).

FIGURE 3

Effect of deglycosylation and membrane environment on nAChR structure. (A) The deconvolved amide I/I′ band from glycosylated (i) and Endo F1 deglycosylated (ii) nAChR reconstituted into soybean asolectin membranes, as well as glycosylated nAChR reconstituted into POPC (iii) membranes. The deconvolved spectra show the frequencies and relative intensities of amide I (1655 cm⁻¹, α-helix and 1639 cm⁻¹, β-sheet) and amide I′ (1645 cm⁻¹, ¹H/²H exchanged α-helix) component bands, and thus provide a sensitive measure of nAChR secondary structure. (B) Nondeconvolved residual amide II band. Note the amide II vibration (1547 cm⁻¹) partially overlaps with broad bands due to side chain asymmetric COO⁻ stretching vibrations near 1580 and 1560 cm⁻¹. Spectra were recorded at 22.5°C after each sample was exchanged for precisely 72 h at 4°C. Each spectrum is representative of between four and seven spectra recorded from each sample.

FIGURE 4

Effect of deglycosylation and membrane environment on the kinetics of nAChR peptide ¹H/²H exchange. (A) Nondeconvolved FTIR spectra of glycosylated (i) and Endo F1 deglycosylated (ii) nAChR reconstituted into asolectin membranes as well as POPC reconstituted nAChR (iii). Spectra were recorded after 3 (solid line, top), 24 (dashed line, middle), and 750 (dotted line, bottom) min of exposure to deuterated buffer at 22.5°C. (B) The hydrogen exchange kinetics are plotted as the ratio of residual amide II to amide I band intensity as a function of time after exposure to ²H₂O T. californica ringer buffer for glycosylated (solid circles, n = 5), Endo F1 deglycosylated (open circles, n = 2), and glycosylated POPC reconstituted nAChR (solid diamonds, n = 4). Error bars represent ± SD of the mean. Where error bars are not visible they are smaller than the data points. (C) Deconvolved lipid ester carbonyl stretching region. Hydrogen-bonded ester carbonyls (1729 cm⁻¹) and nonhydrogen-bonded ester carbonyls (1741 cm⁻¹) are labeled.

FIGURE 5

Effect of deglycosylation and membrane environment on reconstituted nAChR thermal stability. (A) 3D stack plot showing the spectral changes accompanying thermal denaturation of asolectin reconstituted and glycosylated nAChR. Spectra are not ²H₂O subtracted. Note: the relative magnitude of change in intensity at 1681 cm⁻¹ (and 1623 cm⁻¹) for both the deglycosylated asolectin and POPC samples were similar to those shown above. (B) Percent change in intensity at 1681 cm⁻¹ as a function of temperature for asolectin reconstituted and glycosylated (solid circles, n = 5), Endo F1 deglycosylated (open circles, n = 2) and POPC reconstituted (solid diamonds, n = 5) nAChR membranes. Error bars represent ± SD of the mean. Where error bars are not visible they are smaller than the data points.

FIGURE 6

Effect of deglycosylation and membrane environment on nAChR function. FTIR difference spectra showing the vibrational shifts occurring in the nAChR upon carbamylcholine binding and subsequent desensitization. The difference spectra were acquired from glycosylated (i) and Endo F1 deglycosylated (ii) nAChR reconstituted into soybean asolectin membranes, as well as glycosylated nAChR reconstituted into POPC membranes (iii). Each presented difference spectrum is the average of at least 30 individual difference measurements.