HSP60 is transported through the secretory pathway of 3‐MCA‐induced fibrosarcoma tumour cells and undergoes N‐glycosylation

Abstract

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri-and tetraantennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment. Structured digital abstractl Grp94 and Hsp60 colocalize by cosedimentation through density gradient (View interaction) Abbreviations 3-MCA, 3-methylcholantrene; BFA, brefeldin A; EGFA, epidermal growth factor receptor; ER, endoplasmic reticulum; FACS, fluorescenceactivated cell sorting; FGF-2, fibroblast growth factor-2; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; HSP, heat shock protein; MFI, mean fluorescence intensity; NP-HPLC, normal phase-HPLC; TEM, transmission electron microscopy; TLR, Toll-like receptor.

Extracellular Toxoplasma gondii tachyzoites metabolize and incorporate unnatural sugars into cellular proteins

“…The captured and eluted proteins were analyzed via mass spectrometry (MS) and the resulting 89 candidate proteins are listed in Table 1. Importantly, the hits included orthologs of proteins that are known to be O-GlcNAcylated in other species (HSP60, enolase, GAPDH) [49, 50] as well as proteins that were identified in two lectin MS surveys of tachyzoite proteins (myosin A, GAP50) [16, 17]. Some proteins are previously characterized components that are specific to Toxoplasma (SAG1, SAG2) [51, 52] while others are novel hypothetical proteins identified in the Toxoplasma genome.…”

Cytosolic Hsp60 Can Modulate Proteasome Activity in Yeast

“…3) An alternative mechanism involves partially translocated Hsp60 (amino terminus only), which is processed and then reversed translocated back into the cytosol in a process that has been described for a number of proteins including fumarase and aconitase in yeast (36 -38). 4) A subset of Hsp60 is translocated by an unknown process into the ER and is then distributed to various compartments of the cell via the endomembrane system, a surprising mechanism that has been suggested very recently (39). This issue regarding Hsp60 targeting and localization in the cytosol remains to be resolved not only in yeast but also in higher eukaryotes.…”

The dissociation of the Hsp60/pro-Caspase-3 complex by bis(pyridyl)oxadiazole copper complex ( CubipyOXA ) leads to cell death in NCI-H292 cancer cells

“…The function of extracellular Hsp60 is still under discussion. For instance, it may mediate interactions with the immune cells and other body tissues and alter the tumor microenvironment [11,12,55,57].…”