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Drosophila Glycoprotein 93 Is an Ortholog of Mammalian Heat Shock Protein gp96 (grp94, HSP90b1, HSPC4) and Retains Disulfide Bond-Independent Chaperone Function for TLRs and Integrins¹

Crystal Morales,^2* Shuang Wu,^2* Yi Yang,^3* Bing Hao, and Zihai Li^4*

^*Department of Immunology and Department of Microbial, Molecular and Structural Biology, University of Connecticut School of Medicine, Farmington, CT 06030

Mammalian heat shock protein gp96 is an obligate chaperone for multiple integrins and TLRs, the mechanism of which is largely unknown. We have identified gp93 in Drosophila having high sequence homology to gp96. However, no functions were previously attributed to gp93. To determine whether gp93 and gp96 are functionally conserved, we have expressed gp93 in gp96-deficient mouse cells. Remarkably, the Drosophila gp93 is able to chaperone multiple murine gp96 clients including integrins ₄, _L, and β₂ and TLR2 and TLR9. This observation has led us to examine the structural basis of the chaperone function of gp96 by a close comparison between gp96 and gp93. We report that whereas gp96 undergoes intermolecular disulfide bond formation via Cys¹³⁸, gp93 is unable to do so due to the absence of a cysteine near the same region. However, abrogation of disulfide bond formation by substituting C with A (C138A) in gp96 via site-directed mutagenesis did not compromise its chaperone function. Likewise, gp93 chaperone ability could not be improved by forcing intermolecular bond formation between gp93 N termini. We conclude that gp93 is the Drosophila ortholog of gp96 and that the chaperone function of the two molecules is conserved. Moreover, gp96 N-terminal disulfide bond formation is not critical for its function, underscoring the importance of N-terminal dimerization via non-disulfide bond-mediated interactions in client protein folding by gp96. Further study of gp96 from an evolutionary angle shall be informative to uncover the detailed mechanism of its chaperone function of client proteins in the secretory pathway.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by National Institutes of Health Grants CA100191 and AI070603 (to Z.L.).

² C.M. and S.W. contributed equally to this work.

³ Current address: Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University, New York, NY 10016.

⁴ Address correspondence and reprint requests to Dr. Zihai Li, Center for Immunotherapy of Cancer and Infectious Diseases, Department of Immunology, University of Connecticut School of Medicine, MC1601, 263 Farmington Avenue, Farmington, CT 06030-1601. E-mail address: zli{at}up.uchc.edu

⁵ Abbreviations used in this paper: HSP, heat shock protein; ER, endoplasmic reticulum; WT, wild type; RIPA, radioimmunoprecipitation assay; DSP, dithiobis-succinimidyl propionate; HA, hemagglutinin; IB, immunoblot; MFI, mean fluorescence intensity; co-IP, coimmunoprecipitation.