title: Distinct binding sites for the ATPase and substrate-binding domain of human Hsp70 on the cell surface of antigen presenting cells.
abstract: Hsp70 has high potential as an immune-adjuvant molecule: it mediates cytokine expression and maturation of antigen presenting cells (APCs) and also elicits a cytotoxic T-lymphocyte (CTL) response to antigenic peptides. How Hsp70 interacts with APCs is only poorly understood. Various surface proteins have been implicated in binding Hsp70 but their role in antigen presentation has remained controversial. The specific aim of this work was to determine the binding and uptake of human full-length Hsp70 as well as its separate ATPase (N70) and substrate-binding domains (C70) by APCs. Using laser scanning microscopy and FACS analysis, we established the existence of at least two distinct receptors for Hsp70, which are localized to distinct microdomains of the APC membrane. These receptors interact with the N70 and C70 domains of Hsp70, respectively. This observation was supported by the finding of a substantial portion of Hsp70 and C70, but not N70, in a detergent resistant membrane fraction. Accordingly, C70 and N70 did not compete with each other for binding. The bound proteins were rapidly internalized, with N70 and C70 localizing to separate endosomal compartments. Similarly, internalized free and peptide-loaded Hsp70 segregated rapidly within the cell. Efficient cross presentation of antigenic peptide bound to Hsp70 or C70 was demonstrated with the B3Z read out system. Consequently, the interaction of C70 with its putative receptor seems to be responsible for Hsp70-mediated cross presentation. Future studies should make use of C70 in identifying the uptake receptor of Hsp70-peptide complexes. In addition we could observe a stimulation of uptake of free peptide by preincubation with Hsp70 and N70, but not C70, whereas an Hsp-dependent cytokine secretion could not be detected. Consequently, by employing the individual domains it may be possible to distinguish between the different outcomes of Hsp70 treatment, like immune stimulation, DC maturation and antigen-specific responses.
authors.affiliation: Heidelberg University Biochemistry Center (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Zitzler
authors.forename: Alice
authors.initials: A
authors.lastname: Hellwig
authors.forename: Franz-Ulrich
authors.initials: FU
authors.lastname: Hartl
authors.forename: Felix
authors.initials: F
authors.lastname: Wieland
authors.forename: Petra
authors.initials: P
authors.lastname: Diestelkötter-Bachert
mesh_terms: D000251:Adenosine Triphosphatases / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D000251:Adenosine Triphosphatases