Detection of Heat Shock Protein-70 from Trichinella spiralis Larvae Using a Modification of the Routine Western Blotting Procedure

Glutamine (GLN) plays a key role in cellular protection following injury via enhancement of heat shock protein 70 (HSP70). The pathway by which GLN enhances HSP70 is unknown. GLN is a key substrate for the hexosamine biosynthetic pathway (HBP), which has been shown to induce HSP70. We sought to explore the role of the HBP in GLN-mediated HSP70 expression. Both chemical inhibitors and small interfering (si)RNA knockdown of key HBP enzymes were used in mouse embryonic fibroblast cells to determine the effects of the HBP on HSP70 expression. The O-glycosylation, nuclear translocation, and transcriptional activation of heat shock factor-1 (HSF-1) and Sp1 were evaluated using immunoprecipitation, Western blotting, and luciferase assays. HSP70 expression levels were evaluated via ELISA and Western blotting. GLN augmented HBP activity before and after heat stress (HS). Chemical inhibition of HBP enzymes reduced GLN-mediated HSP70 expression. Specific siRNA targeting of the key HBP enzyme UDP- N-acetylglucosamine (GlcNAc): polypeptide- O-β-acetylglucosaminyltransferase (OGT) blocked GLN-mediated HSP70 expression and attenuated GLN-mediated cellular protection post-HS. Chemical and siRNA attenuation of the HBP blocked GLN-induced nuclear translocation of Sp1 and HSF-1, which are key to maximal HSP70 expression. Finally, immunoprecipitation revealed HSF-1 was O-glycosylated, and GLN enhanced this effect. These results suggest that metabolism of GLN via the HBP enhances HSP70 expression. This effect appears to be mediated via O-glycosylation, nuclear translocation, and transcriptional activation of Sp1 and HSF-1. This is an important mechanistic description of a pathway that appears responsible for GLN-mediated HSP70 expression.

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Molecular cloning and characterization of heat shock protein 70 from Trichinella spiralis

Acta Tropica ◽

10.1016/j.actatropica.2009.01.003 ◽

2009 ◽

Vol 110 (1) ◽

pp. 46-51 ◽

Cited By ~ 35

Author(s):

Shaohua Wang ◽

Xinping Zhu ◽

Yaping Yang ◽

Jing Yang ◽

Yuan Gu ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Molecular Cloning ◽

Heat Shock Protein 70 ◽

Trichinella Spiralis

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Detection of Humoral Response Using a Recombinant Heat Shock Protein 70, DnaK, of Mycoplasma haemofelis in Experimentally and Naturally Hemoplasma-Infected Cats

Clinical and Vaccine Immunology ◽

10.1128/cvi.00320-10 ◽

2010 ◽

Vol 17 (12) ◽

pp. 1926-1932 ◽

Cited By ~ 17

Author(s):

Emily N. Barker ◽

Chris R. Helps ◽

Kate J. Heesom ◽

Christopher J. Arthur ◽

Iain R. Peters ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Western Blotting ◽

Heat Shock Protein 70 ◽

Acute Infection ◽

Amino Acid Sequences ◽

Mass Spectrometry Data ◽

Clinical Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Two Dimensional Gel Electrophoresis

ABSTRACT Hemoplasmas is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis is the most pathogenic, while “Candidatus Mycoplasma haemominutum” and “Candidatus Mycoplasma turicensis” are less pathogenic. Shotgun libraries of fragmented M. haemofelis genomic DNA were constructed, and random colonies were selected for DNA sequencing. In silico-translated amino acid sequences of putative open reading frames were compared to mass spectrometry data from M. haemofelis protein spots identified as being immunogenic by two-dimensional gel electrophoresis and Western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (DnaK) homolog, elongation factor Ts, and a fragment of phosphoglycerate kinase found during library screening. A full-length copy of the M. haemofelis dnaK gene was cloned into Escherichia coli and recombinantly expressed. Recombinant M. haemofelis DnaK was purified and then used in Western blotting and an enzyme-linked immunosorbent assay (ELISA) to investigate the humoral immune response during acute infection in cats experimentally infected with M. haemofelis, “Ca. Mycoplasma haemominutum,” or “Ca. Mycoplasma turicensis”. The recombinant M. haemofelis DnaK ELISA also was used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n = 254). Experimentally infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with M. haemofelis than those seen in cats inoculated with “Ca. Mycoplasma haemominutum” or “Ca. Mycoplasma turicensis,” by both Western blotting and ELISA. Of the clinical samples, 31.1% had antibodies detected by the ELISA but only 9.8% were positive by PCR for one or more hemoplasmas.

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