Differential Glycosylation and Modulation of Camel and Human HSP Isoforms in Response to Thermal and Hypoxic Stresses

Abstract

Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances.

Keywords: CRYAB; HSPA6; O-GlcNAc; camel; heat shock proteins; heat stress; hypoxia; protein expression.

Figure A1

The cDNA sequence of camel HSPA6 (accession number, MG021195). The upper lines represent the cDNA sequence while the lower lines refer to the deduced amino acids.

Figure A2

Expression and mass spectrometry analysis of camel and human HSPA6. (A) 9% SDS showing the expressed protein forms of recombinant camel HSPA6 (cHSPA6) and human HSPA6 (hHSPA6) after transfection of COS-1 cells and IP using anti-Myc antibody. The cHSPA6 revealed 2 isoforms (a,b) while hHSPA6 showed 3 isoforms (c–e); (B) Mass Spectrometry results of the excised cHSPA6 and hHSPA6 protein bands. The green coloration indicates the coverage of the analyzed peptides.

Figure A3

(A) cHSPA6 and hHSPA6 transiently expressed in COS-1 cells were subjected to Helix pomatia Sepharose as described previously. The lectin-bound material was immunoprecipitated with anti-Myc and analyzed by Western blotting. hHSPA6 did not bind to the lectin in contrast to cHSPA6 (left panel). The right panel shows an overexposed version of the same blot that also shows that no binding of hHSPA6 to the lectin has occurred; (B) The lectin pull down assay was repeated under different temperatures as indicated (left panel). No O-GlcNAc modified forms of hHSPA6 were detected in contrast to cHSPA6 that bound to the lectin. The right panel shows Western blot of the corresponding total lysates as control; (C) Similar to (B), the same lectin experiment was performed after different oxygen treatments. Here again no O-GlcNAc forms of hHSPA6 in contrast to cHSPA6 that revealed O-GlcNAc forms under both hypoxic and normoxic conditions. The Western blot of the corresponding lysates is shown at the right panel as control.

Figure 1

Homology analysis of the deduced amino acid sequences of cDNA clones of camel and human HSPs. Sequence alignment was produced by Clustal Omega and edited using GeneDoc version 2.7.000. (A) Amino acid alignment of camel and human CRYAB showing the four different amino acids shaded in black; (B) Alignment of isolated camel HSPA6 (GenBank, MG021195) with reported camel HSPA6 sequence (GenBank, ADO12067.1) showing amino acid variations at five amino acids as indicated by the black shaded positions; (C) Amino acid alignment of isolated camel HSPA6 (GenBank, MG021195) and human HSPA6 showing variant amino acids shaded in black. An asterisk indicates a number of ten amino acid residues while dots in the lower lines designate identical amino acid residues to the corresponding upper ones.

Figure 1

Homology analysis of the deduced amino acid sequences of cDNA clones of camel and human HSPs. Sequence alignment was produced by Clustal Omega and edited using GeneDoc version 2.7.000. (A) Amino acid alignment of camel and human CRYAB showing the four different amino acids shaded in black; (B) Alignment of isolated camel HSPA6 (GenBank, MG021195) with reported camel HSPA6 sequence (GenBank, ADO12067.1) showing amino acid variations at five amino acids as indicated by the black shaded positions; (C) Amino acid alignment of isolated camel HSPA6 (GenBank, MG021195) and human HSPA6 showing variant amino acids shaded in black. An asterisk indicates a number of ten amino acid residues while dots in the lower lines designate identical amino acid residues to the corresponding upper ones.

Figure 2

Cloning, expression and localization of Camelus dromedarius and human HSPs: (A) Schematic diagram showing the general design of cloned camel and human HSPs. Camel and human CRYAB or HSPA6 are Myc-tagged in their C-terminal and their expression is controlled by cytomegalovirus (CMV) promotor; (B) The upper and middle panels show Camelus dromedarius and human CRYAB as well as HSPA6 amplified cDNA products with sizes of 1932 bp and 528 bp, respectively. Amplified GAPDH cDNA product in the lower panel was used as control for RNA isolation and cDNA synthesis; (C,D) Western blot analysis of proteins analyzed by SDS-PAGE on 9% slab gels showing expression of camel and human CRYAB and HSPA6 respectively after transfection in COS-1 cells. Camel and human CRYAB were detected as protein bands of an apparent molecular weight of 25 kDa while camel and human HSPA6 specific bands run at around 70 kDa on the gel. Non-transfected COS-1 cells (NT). Actin, a house keeping protein was used as a loading control; (E) Immunoprecipitation of camel and human HSPA6 showing different isoforms between the two HSPs upon expression in COS-1 cells. As illustrated, camel HSPA6 (dashed arrows) has two isoforms, a highly expressed high molecular weight form and a lower molecular weight faint one while the human HSPA6 (continuous arrows) reveals three protein bands with the lowest being the most expressed isoform; (F) Immunoprecipitation of ³⁵S labeled Myc tagged camel and human HSPA6 confirms the results in C; (G) Laser confocal microscopy reveals camel and human HSPA6 and CRYAB in the cytosol of transfected COS-1 cells. HSP (green) was visualized together with the cell nucleus (blue) and calnexin (red) as an ER marker. Scale bars: 25 μm or 10 μm.

Figure 3

Differential glycosylation of camel and human HSPA6. (A) Helix pomatia (lectin)-conjugated Sepharose was used to discriminate between the glycan structures of the Myc-tagged camel and human HSPA6 proteins. The extracted proteins from camel/human HSPA6-transfected COS-1 cells were mixed with Helix pomatia lectin-Sepharose for 2–3 h and the bound proteins were eluted with 10 mM N-acetyl-glucosamine. Camel HSPA6, but not the human species bound to the lectin. As a positive control, immunoprecipitation (IP) of camel and human HSPA6 was performed; (B) Similar experiment was performed for the Myc-tagged camel and human CRYAB. In contrast to camel HSPA6, both chaperones did not bind to the lectin. HSPA6 and CRYAB were detected by anti-Myc antibody. The Western blot shown is representative of three experimental independent repeats.

Figure 4

Variable response of camel and human HSPA6 isoforms to different temperatures. (A) Western blots of immunoprecipitated camel (C) or human (H) HSPA6 proteins from transfected COS-1 cells and exposure to different temperatures (39.5 °C, 37 °C and 20 °C); (B) The loading controls for (A) utilized equal amount of proteins in lysates of transfected COS-1 cells; (C,D) Quantification of each camel/human HSPA6 isoform in the total protein isoforms. Camel HSPA6 is identified by two isoforms (higher (cH) and lower (cL)) while human HSPA6 exhibits three isoforms (higher (hH), middle (hM) and lower (hL)). Statistically significant values are denoted by an asterisk and the blots are representative data sets out of five independent repeats.

Figure 5

Response of camel and human HSPA6 isoforms to different oxygen concentrations. (A) 24 h after transfection, cells were cultured under normal or 1% oxygen levels for 15 h followed by immunoprecipitation of HSPA6 and Western blotting; (B) The loading controls for (A) utilized equal amount of proteins in lysates of transfected COS-1 cells; (C,D) Quantification of each camel or human HSPA6 isoform in the total protein isoforms after incubation under hypoxic or normoxic conditions. As described in Figure 4, camel HSPA6 is identified by two isoforms (higher (cH) and lower (cL)), while human HSPA6 exhibits three isoforms (higher (hH), middle (hM) and lower (hL)). Statistically significant values are denoted by an asterisk and the blots are representative data sets out of five independent repeats.