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O-GalNAcylation of RANTES Improves Its Properties as an HIV-1 Entry Inhibitor Xiaoyang Guan, Patrick K Chaffey, Huan Chen, Wei Feng, Xiuli Wei, LiuMeng Yang, Yuan Ruan, Xinfeng Wang, Yaohao Li, Kimberly B Barosh, Amy H Tran, Jaimie Zhu, Wei Liang, Yong-Tang Zheng, Xu Wang, and Zhongping Tan Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00875 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017
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Biochemistry
O-GalNAcylation of RANTES Improves Its Properties as an HIV-1 Entry Inhibitor Xiaoyang Guan,1, ‡ Patrick K. Chaffey,1, ‡ Huan Chen,2, ‡ Wei Feng,3, ‡ Xiuli Wei,4 Liu-Meng Yang,2 Yuan Ruan,1 Xinfeng Wang,1 Yaohao Li,1 Kimberly B. Barosh,1 Amy H. Tran,1 Jaimie Zhu,1 Wei Liang,4 Yong-Tang Zheng,2,* Xu Wang,3,* and Zhongping Tan1,* 1
Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder CO 80303, United States 2 Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China 3 Department of Chemistry & Biochemistry, Arizona State University, Tempe, AZ 85287, United States 4 Protein & Peptide Pharmaceutical Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China ABSTRACT: Many human proteins have the potential to be developed as therapeutic agents. However, side-effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Posttranslational modifications (PTMs) can improve protein properties. But due to significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side-effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco-variant, a truncated RANTES containing a disaccharide Galβ1-4GalNAc α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also low inflammatory properties. Moreover, our results provided a structural basis for the observed changes on RANTES’ properties. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.
INTRODUCTION Human proteins are an important source of therapeutic agents.1 Compared to small molecules, they have some clear advantages. They are generally very selective and potent, predictably metabolized, and tend to be well tolerated in patients.2,3 However, while many proteins have the potential to be used as highly effective medical treatments for a wide range of diseases, side-effects have limited their therapeutic applications.4,5 For these reasons, there remain many proteins with significant therapeutic potential that have not been successfully developed into clinical stage drugs despite many years of sustained effort. Previous research has established several feasible ways of overcoming side-effects of protein drugs, including posttranslational modifications (PTMs) like glycosylation.6-8 Tweaking the glycosylation pattern and/or glycan structure of proteins with an aim to control the physical properties or function of that resulting glycoprotein is commonly known as glycoengineering; and earlier work suggests that conformationally flexible regions which are also important for biological
function tend to be fruitful regions for glycoengineering investigations.9,10 However, despite the promise of glycoengineering, the full potential of protein glycosylation as a handle for controlling both the biological activity and physical shortcomings of protein-based therapeutics has not been realized. In particular, neither the scope of possible improvements nor the extent to which such improvements might be generally applied have been firmly established.11 This is primarily because of our poor understanding of structure-property relationships for glycoproteins, which in turn is largely due to the difficulties of obtaining collections of pure, homogeneous glycoforms carrying systematically varied and wellcharacterized glycan structures. Here, we apply a chemical glycobiology approach to obtain detailed insights into the effects of glycosylation on protein properties;12 with our focus being on a small representative therapeutic protein, RANTES (Regulated on activation, normal T cell expressed and secreted, also known as CCL5). This research takes advantage of recent advances in chemical synthesis, which have opened practicable synthetic routes to gly-
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coproteins of up to ~200 amino acid residues, an upper limit which conveniently covers many soluble human proteins.13,14 In this study, we intended to uncover whether O-glycosylation and other PTMs are important for improving RANTES for human immunodeficiency virus type 1 (HIV-1) entry inhibition.15,16
Figure 1. The mechanism of RANTES inhibition of the HIV-1 envelope complex mediated viral binding to host cells.
HIV continues to be a major global public health issue that currently affects approximately 36.7 million people worldwide and about 35 million people have died of acquired immune deficiency syndrome (AIDS)-related illnesses since the early 1980s.17,18 Most HIV infections belong to the HIV-1 subtype, with HIV-2 being mostly confined to west Africa.19 HIV-1 infection begins when virus particles bind and penetrate the cell membrane through a several-step process: the virus first binds its primary receptor on the host cell membrane, cluster of differentiation 4 (CD4), which induces conformational changes in the viral glycoprotein 120 (gp120) that then allow binding to one of two co-receptors: C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) (Fig. 1).16,17 With receptor and co-receptor bound, membrane fusion occurs depositing the viral genome inside the host cell.20 Research shows that virus particles responsible for the initial infection in individuals use only CCR5 and later on in the infection, during the AIDS stage, CXCR4-utilizing strains emerge.21 Currently, there is no effective cure for HIV-1/AIDS. In order to develop treatment and prevention strategies for HIV-1 infection, a great deal of effort has been focused on disrupting the interactions between the virus and either CD4, CCR5, or CXCR4, since they are absolutely required for HIV-1 infection.18 Also important is that by acting early and outside the cell, blocking CD4, CCR5 and/or CXCR4 offers advantages over inhibiting the late stage processes like reverse transcription and DNA integration.16 Unlike CD4 and CXCR4, CCR5 is not essential for normal human function,22 and thus blocking the interaction of HIV-1 and CCR5 appears to be a promising strategy to prevent or limit viral entry. Currently, the only CCR5-targeting, FDA-approved HIV-1 entry inhibitor, maraviroc (MAV), is a small molecule that likely blocks HIV-1 through an allosteric, noncompetitive mechanism.23,24 As the natural ligand for CCR5, RANTES has been shown to inhibit HIV-1 entry (Fig. 1).15 Although the exact mechanism for this viral inhibition is still not fully understood, it is thought to occur by a combination of competitive binding and ligand-induced receptor endocytosis.25 These differences in mechanisms of action between RANTES and MAV raise the very intriguing possibility that RANTES analogs could be an effective strategy to deal with the emerging MAV resistance.24,26,27 Also important is that RANTES-based molecules could have the additional benefit of stimulating CCR5
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internalization and long-term sequestration.28 Together, these factors make the development of RANTES-based HIV-1 entry inhibitors an attractive research opportunity. Unfortunately, RANTES signaling through CCR5 functions to stimulate the migration (chemotaxis) of leukocytes, and prolonged administration of RANTES can lead to uncontrolled accumulation of leukocytes in tissues, detrimental inflammation, and severe tissue damage.16,29,30 This side-effect has significantly limited the therapeutic application of RANTES, especially in the context of topical inhibitors of HIV-1 infection because inflammation of mucosal membranes, a common occurrence upon application of topical RANTES-based inhibitors, is known to enhance HIV-1 infectivity.31 In order to use RANTES as a successful HIV-1 entry inhibitor, it is necessary to decrease its pro-inflammatory activity while also largely maintaining its ability to inhibit viral entry. While this goal is clear, achieving it has been a challenge.16,3234 Since post-translational modifications (PTMs) influence both the biophysical and biological properties of proteins, one promising approach is to use PTMs as tools to minimize unwanted inflammatory activities of RANTES.18,25,35-41 Naturally occurring RANTES is post-translationally modified in a few different ways, including truncation at the N-terminus (deletion of one, two, or three amino acids from the N-terminus of RANTES), O-glycosylation at Ser4 and Ser5, and oxidation at Met67 (Fig. 2).42 Like most glycoproteins, the precise chemical structures of glycans on RANTES have not been elucidated. The fact that O-linked glycans on human serum proteins like RANTES are predominantly of the mucin-type43 and the fact that all mucin-type O-glycans are enzymatically biosynthesized in a stepwise manner from a GalNAcα1-O-Ser/Thr precursor44 suggest that core 1 and/or core 2 O-glycans, which may be smaller or larger than the ones shown in Figure 2, should be present on Ser4 and Ser5.45 The abundance of glycosylated RANTES may be low (
