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Grp94, Mammalian Heat Shock Pr Drosophila Glycoprotein 93 Is an Ortholog of Mammalian Heat Shock Protein gp96 (grp94, HSP90b1, HSPC4) and Retains Disulfide Bond-Independent Chaperone Function for This information is current as TLRs and Integrins of October 3, 2021. Crystal Morales, Shuang Wu, Yi Yang, Bing Hao and Zihai Li J Immunol 2009; 183:5121-5128; Prepublished online 28 September 2009; Downloaded from doi: 10.4049/jimmunol.0900811 http://www.jimmunol.org/content/183/8/5121 References This article cites 46 articles, 8 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/183/8/5121.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 3, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 Li4* 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 ␤ Downloaded from 2 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 http://www.jimmunol.org/ 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 Journal of Immunology, 2009, 183: 5121–5128. he glycoprotein gp96 (1), known also as heat shock pro- essential functions in chaperoning multiple TLRs, including tein (HSP)5 90b1 (2), grp94 (3), endoplasmic reticulum TLR1, TLR2, TLR4, TLR5, TLR7, and TLR9, and a variety of (ER) p99 (4), endoplasmin (5), and HSPC4 (6), is the ER integrins such as ␣ , ␣ , and ␤ (13–15). Similar to HSP90, gp96 T 4 L 2 paralog of the cytosolic chaperone HSP90 (7). gp96 is thought to can hydrolyze ATP (16–18), and it is thought to function as a play general roles in both the unfolded protein response in the ER homodimer (18). It has an N-terminal signal peptide, followed se- by guest on October 3, 2021 and the ER-associated degradation pathway to maintain protein quentially by an ATPase domain, a charged middle domain, a homeostasis (8, 9). Up to 10% of the cytosolic proteins can be dimerization domain, and the C-terminal KDEL ER retention sig- chaperoned by HSP90 (10, 11). By comparison, the number of nal. Deletion of either the ATPase or the C-terminal dimerization confirmed client proteins of gp96 is limited. Although Ig is among domain results in total loss of chaperone function, whereas re- the first implicated clients of gp96 (12), a recent study demon- moval of the middle charged domain or C-terminal ER retention strates that assembly and production of Abs are uncompromised in signal does not alter its chaperone function (14). Although the the absence of gp96 (13). Importantly, gp96 has been found to play exact mode of the chaperone function of gp96 is unclear, it is thought that the binding of gp96 to client proteins leads to trans- formation of the low-energy-state open conformation of a gp96 to *Department of Immunology and †Department of Microbial, Molecular and Structural a closed conformation through N-terminal dimerization and final Biology, University of Connecticut School of Medicine, Farmington, CT 06030 maturation of client protein folding (18). Received for publication March 12, 2009. Accepted for publication August 5, 2009. The two major clients of gp96, integrins and TLRs, are evolu- The costs of publication of this article were defrayed in part by the payment of page tionarily conserved. Restricted to all species of metazoan (19), charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. integrins are transmembrane proteins that link the extracellular matrix to the cytoskeleton and are important for various biological 1 This work was supported in part by National Institutes of Health Grants CA100191 and AI070603 (to Z.L.). processes such as survival, proliferation, apoptosis, adhesion, che- 2 C.M. and S.W. contributed equally to this work. motaxis, polarity, differentiation, and gene expression (20, 21). ␣ ␤ 3 Current address: Kimmel Center for Biology and Medicine, Skirball Institute of Integrins consist of and subunits which heterodimerize to form Biomolecular Medicine, New York University, New York, NY 10016. the receptor (22). Humans and mice have 18 ␣ and 8 ␤ subunits, 4 Address correspondence and reprint requests to Dr. Zihai Li, Center for Immuno- which form 24 heterodimers and can be grouped as collagen re- therapy of Cancer and Infectious Diseases, Department of Immunology, University of ceptors, laminin receptors, RGD receptors, and leukocyte receptors Connecticut School of Medicine, MC1601, 263 Farmington Avenue, Farmington, CT 06030-1601. E-mail address: [email protected] (19, 20, 23). They are capable of inside-out signaling as well as 5 Abbreviations used in this paper: HSP, heat shock protein; ER, endoplasmic retic- outside-in signaling (19, 20), and many are targeted by both vi- ulum; WT, wild type; RIPA, radioimmunoprecipitation assay; DSP, dithiobis-succin- ruses and bacteria as receptors (19). imidyl propionate; HA, hemagglutinin; IB, immunoblot; MFI, mean fluorescence in- Like integrins, TLRs are also type I transmembrane receptors. tensity; co-IP, coimmunoprecipitation. TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, TLR11, and TLR12 Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 are present at the cell surface; whereas TLR3, TLR7, TLR8, and www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900811 5122 FOLDING TLRs AND INTEGRINS BY Drosophila gp93 TLR9 reside in the acidic endosomes. All TLRs are believed to ER retention signal to enable efficient ER targeting in a murine system, homodimerize to function except TLR1 and TLR6 which need to and the Flag epitope (DYKDDDDK) was placed immediately before the A137C heterodimerize with TLR2 (24). These receptors recognize bacte- KDEL sequence (37). To generate the gp93 mutant, the following primers were designed with Stratagene’s online primer design program: rial and viral constituents, and the resulting signaling cascade ac- gp93A137Cs, 5Ј-ctgcacatccgcattaagtgcgacaaggagaacaagg-3Ј; gp93A137Cas tivates the innate immune system. Exhibiting high similarity to the 5Ј-ccttgttctccttgtcgcacttaatgcggatgtgcag-3Ј. Using gp93 as a template, the IL-1/IL-18 receptor family, TLRs have Toll/IL-1R domains in mutation was incorporated with the aid of the QuikChange II XL Site- their cytoplasmic tails that are important for recruiting the down- Directed Mutagenesis Kit (Stratagene) per manufacturer’s protocol. It was later amplified by PCR with the same primers used to create gp93Flag. The stream signaling adaptor molecules TIRAP, MyD88, TRAM, and C3A and C5A mutants of gp96 were also generated using the same mu- TRIF (24). These pathways culminate in activation of the tran- tagenesis kit. The mutant sequence necessary to create the desired cysteine scription factors NF-␬B and IFN regulatory factor-3, and the re- to alanine substitution was incorporated into these primers. All cloned sulting production of type 1 IFN and inflammatory cytokines such genes of interests were verified by sequencing. as IL-6. Although many of the same signaling molecules are in- Cells volved, differential activation of TLRs can lead to different re- sponses (24). Recent studies indicate that the ectodomain of
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