Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2019
Elucidating the role of N-glycosylation in the interaction between Fc γ Receptors (FcγRs) and Immunoglobulin G (IgG) through characterization of endogenous FcγR N-glycosylation and FcγRIII (CD16) binding
Jacob Timothy Roberts Iowa State University
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Recommended Citation Roberts, Jacob Timothy, "Elucidating the role of N-glycosylation in the interaction between Fc γ Receptors (FcγRs) and Immunoglobulin G (IgG) through characterization of endogenous FcγR N-glycosylation and FcγRIII (CD16) binding" (2019). Graduate Theses and Dissertations. 17771. https://lib.dr.iastate.edu/etd/17771
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Elucidating the role of N-glycosylation in the interaction between Fc Receptors (FcRs) and Immunoglobulin G (IgG) through characterization of endogenous FcR N-glycosylation and FcRIII (CD16) binding
by
Jacob Timothy Roberts
A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY
Major: Biochemistry
Program of Study Committee: Adam W. Barb, Co-major professor Richard Honzatko, Co-major professor Amy Andreotti Douglas Jones Eric Underbakke
The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2019
Copyright © Jacob Timothy Roberts, 2019. All rights reserved. ii
DEDICATION
To my wife, Angela, and two daughters, Savannah and Lillian
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………………………………v
ABSTRACT……………………………………………………………………………………...vi
CHAPTER 1. INTRODUCTION. MULTIPLE VARIABLES AT THE LEUKOCYTE CELL SURFACE IMPACT FC RECEPTOR-DEPENDENT MECHANISMS…………....1 Introduction: The importance of modulating the Fc-FcR interaction……………...1 Receptor presentation at the cell surface…………………………………………….3 Variability in receptor amount………………………………………………………….4 Receptor clustering at the membrane is required for effector function……………7 The type of FcR membrane anchor impacts activation…………………………..12 N-linked glycosylation heterogeneity depends on multiple factors……………….13 N-linked glycosylation of antibody affects FcRIII (CD16) binding……………….16 CD16a N-glycosylation composition affects binding to IgG1……………………..16 Works cited……………………………………………………………………………..18
CHAPTER 2. A SINGLE AMINO ACID DISTORTS THE FC RECEPTOR IIIB / CD16B STRUCTURE UPON BINDING IMMUNOGLOBULIN G1 AND REDUCES AFFINITY RELATIVE TO CD16A…………………………………………………………....29 Abstract…………………………………………………………………………………29 Introduction……………………………………………………………………………..30 Results………………………………………………………………………………….34 Discussion………………………………………………………………………………46 Experimental Procedures……………………………………………………………..49 Works Cited…………………………………………………………………………….52
CHAPTER 3. SITE-SPECIFIC N-GLYCAN ANALYSIS OF ANTIBODY-BINDING FC RECEPTORS FROM PRIMARY HUMAN MONOCYTES………………………………57 Abstract…………………………………………………………………………………57 Introduction……………………………………………………………………………..58 Experimental Procedures……………………………………………………………..61 Results………………………………………………………………………………….69 Discussion………………………………………………………………………………80 Works Cited…………………………………………………………………………….83
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CHAPTER 4. RESTRICTED PROCESSING OF CD16A / FC RECEPTOR IIIA N- GLYCANS FROM PRIMARY HUMAN NK CELLS IMPACTS STRUCTURE AND FUNCTION……………………………………………………………………………………..89 Abstract…………………………………………………………………………………89 Introduction……………………………………………………………………………..90 Results………………………………………………………………………………….94 Discussion…………………………………………………………………………….108 Experimental procedures……………………………………………………………111 Works cited……………………………………………………………………………121
CHAPTER 5. GENERAL CONCLUSION………………………………………………….127
APPENDIX A. A RARE ALLOTYPE ON CD16A FROM MONOCYTES ISOLATED FROM PLATELET APHERESIS DONORS AFFECT N-GLYCOSYLATION COMPOSITION AT SITE N45………………………………………………………………128
APPENDIX B. INTRADOMAIN INTERACTIONS IN AN NMDA RECEPTOR FRAGMENT MEDIATE N-GLYCAN PROCESSING AND CONFORMATIONAL SAMPLING …………………………………………………………………………………...134
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ACKNOWLEDGEMENTS
I would like to thank my parents Tim, Lisa and Criss for always pushing me to be better and encouraging me to pursue an education. Also, for providing love and support to pursue my interests fully.
My wife, Angela, for always being there to support me through stressful situations and demanding my best.
My daughters Lily and Savannah for making my life full of excitement and being a constant reminder that I need to lead by example.
Adam Barb for mentoring me through these past 4 years. He made life in graduate school enjoyable while also teaching me how to do be a good scientist. I have achieved tremendous professional growth in these past years working with him and I believe he has been a big part of that. I also appreciate his mentoring as something to emulate in the future.
I would also like to thank my committee members Amy Andreotti, Rich Honzatko,
Eric Underbakke and Doug Jones for always maintaining open lines of communication with me and their constant support of my professional development.
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ABSTRACT
N-glycosylation is one of the most abundant post-translational modifications in humans. N-glycans are processed in the ER and Golgi network by enzymes yielding heterogenous populations of N-glycosylation structures in vivo. N-glycosylation is part of the protein quality control and folding pathways. N-glycans also function in antigenicity, serum half-life, proteolysis protection, protein function and much more. N-glycosylation composition differences have been linked to disease states, age, gender, and pregnancy. These functionally relevant composition differences have also been taken advantage of by the pharmaceutical industry.
Monoclonal antibody therapies (mAb) are estimated to exceed $140B in annual sales by 2024. mAb therapies function through direct binding of molecules in inflammation, neutralization or the complement cascade though the majority require
FcR-mediated cell effector functions including antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular phagocytosis (ADCC). A second generation of glycoengineered monoclonal antibody (mAb) therapies have recently out- performed their counterparts in clinical studies. This efficacy is thought to be directly related to the enhanced binding of antibody for FcRIII (CD16). CD16a is expressed predominantly on NK cells and monocytes and believed to be the main receptor engaged in ADCC. The role of the IgG antibody N-glycan has been well studied by others as well as previous members of the lab. However, the role of FcR N- glycosylation including CD16a, CD16b and CD32a have not been well studied. Surface plasmon resonance (SPR) experiments designed to study the role of FcR N- vii glycosylation have shown less processed, oligomannose and hybrid-type N-glycans bind with higher affinity to IgG1 Fc than with complex-type N-glycans. CD16a expressed in a HEK293 Gnt1(-/-) cells give predominant Man5 N-glycosylation and bind 12-50 fold tighter than complex-type while CD16a with hybrid and oligomannose (Man9) bound 2-4 fold tighter than complex-type. Interestingly, when all FcRs were expressed with Man5
N-glycans instead of complex-type an enhanced affinity for IgG1 was observed on one
FcR, CD16a, and is specific to one of five N-glycosylations sites, the N162 N-glycan.
CD16b is a nearly identical paralogue to CD16a and differs by only four residues in the extracellular binding domains, thus it is a surprise that CD16b has a much weaker affinity for IgG1 and does not share an enhanced affinity when expressing Man5 as described with CD16a. A better understanding for this could lead to optimal mAb therapies. Interestingly only residue 129, a glycine in CD16a and aspartate in CD16b is the only residue of the four within the binding interface. CD16a G129D and CD16b
D129G mutants expressing Man5 or complex-type N-glycans were subjected to SPR binding studies to IgG1 Fc. CD16b D129G bound with a 2-fold higher affinity than
CD16a WT and a 90-fold higher affinity than CD16b WT. The reverse mutation in
CD16a G129D, bound with a 128-fold decreased affinity to CD16a wt and comparable to CD16b WT. Furthermore, when comparing CD16 Man5 vs. complex-type N-glycans, the ‘lec effect’ was only observed in CD16a WT and CD16b D129G suggesting the interaction is more sensitive to CD16 N-glycosylation when G129 is present because when D129 was present there was a minimal difference between CD16 binding to IgG with a Man5 or complex-type N-glycan. An x-ray crystal structure of glycosylated CD16b in complex with afucosylated IgG1 Fc was determined to 2.2 Å resolution along with a viii
250 ns all-atom molecular dynamics simulation showed the D129 forces an unfavorable buckle in the CD16b backbone upon binding to IgG1 Fc. Furthermore, the D129 residue forces R155 to make different contacts with the N162 N-glycan. This may force the
N162 N-glycan into different conformations which are less sensitive to N-glycan composition on CD16b as compared to CD16a. Overall, these results determine D129 is responsible for the lower affinity of CD16b as compared to CD16a and this finding suggests mAb therapies could design antibodies to accommodate this residue.
CD16a N-glycosylation composition affects binding to IgG1 in vitro. This finding lead our group to question whether immune response is modulated through CD16a N- glycan composition in vivo. However, N-glycosylation composition of CD16a from endogenous tissue has not been elucidated before. Our lab developed methods to isolate CD16a from NK cells and monocytes from apheresis filters after platelet and plasma donations. Immunoprecipitation of CD16a typically yielded 1 to 2 g of CD16a from NK cells and 100 ng to 1 g from monocytes. A glycomics approach to CD16a from NK cells found higher oligomannose, less processed, material than what was found in recombinant sources. This study indicated recombinant CD16a glycoforms used by laboratories and the pharmaceutical industry do not recapitulate the material found in humans. A glycoproteomics approach was then used to identify composition of the 5 and 2 N-glycosylation sites of CD16a and CD32a from monocytes. CD16a at
N162 was predominantly complex-type biantennerary, and some donors displayed up to
20% of hybrid-types. N38 and N74 displayed highly processed, complex-type biantennerary. N169 displayed predominantly complex-type biantennerary with some highly processed complex-type. CD16a N45 was predominantly under processed with ix mostly oligomannose and hybrid type. Interestingly, CD16a N45 from monocytes displays more oligomannose than from NK cells. CD16a from NK cells also displayed higher abundance of oligomannose and hybrid-types than CD16a from monocytes indicating higher affinity glycoforms on some NK cell donors. CD32a N64 and N145 from monocytes displayed complex-type biantennerary with two sialic acids unlike any of the CD16a N-glycoyslation sites. Overall, N-glycosylation of CD16a appears to differ by cell-type, donor and site. These methods can now be used to expand donor populations and has the potential to characterize disease-relevant N-glycosylation differences as already has been elucidated in antibodies from populations with various disease states, gender and age. 1
CHAPTER 1. INTRODUCTION. MULTIPLE VARIABLES AT THE LEUKOCYTE CELL SURFACE IMPACT FC RECEPTOR-DEPENDENT MECHANISMS
Portions of this chapter were published as a review in Frontiers in Immunology. DOI: 10.3389/fimmu.2019.00223 by Jacob T. Roberts, Kashyap R. Patel, Adam W. Barb
Initials at the end of the subheading titles indicate contributions to that section
Introduction: The importance of modulating the Fc-FcR interaction (KRP, JTR)
Immunoglobulin G (IgG) is the most thoroughly studied and well characterized molecule of the humoral immune response. IgG activates the immune system through cell-bound Fc Receptors (FcRs; Figure 1.1). The IgG fragment antigen-binding (Fab) domains confer specificity and affinity towards an antigen while the distinct hinge and fragment crystallizable (Fc) domain of the four IgG subclasses (IgG1-4) provide the structural basis for specificity and affinity to bind FcRs (1). The six structurally distinct members of the classical human FcRs (FcRI or CD64, FcRIIa / CD32a, FcRIIb /
CD32b, FcRIIc / CD32c, FcRIIIa / CD16a and FcRIIIb / CD16b) are expressed on leukocytes of both the myeloid and lymphoid lineage. This group of proteins can be divided into two types: activating receptors (CD64, CD32a, CD32c, CD16a and CD16b) that lead to cell activation through immunoreceptor tyrosine-based activation motifs
(ITAM) on cytosolic tails or on co-receptor molecules, and an inhibitory receptor
(CD32b) that signals through immunoreceptor tyrosine-based inhibitory motifs (ITIM) (2- 2
4). Only CD32s contain ITAM or ITIM domains, and the other receptors must associate with an ITAM-containing adaptor protein (FcRI chain or CD3 chain) (3,5). In either situation, the ratio of activating to inhibiting signals determines the outcome of an immune response (6).
Figure 1.1. Multiple variables affect FcR-mediated immune function (JTR, KRP). (A) Cellular variables influencing FcR activity that are present before the effector cell engages a target cell. (B) Cellular variables influencing FcR-mediated activity while the effector cell is engaged with a target cell. (C) Molecular variables associated with the FcRs, antibody and antigen.
Receptor clustering is essential for FcR signaling. Circulating IgG coats an antigen to form an oligomeric complex, positioning the Fc portions of the IgG molecules away from the target surface and exposed to interact with FcRs. The antibody-coated 3 target is also referred to as an immune complex. The multiple IgG molecules of the immune complex provide an opportunity for multivalent interactions with FcR- expressing leukocytes and must compete with non-complexed serum antibodies occupying the FcRs that will, in turn, cluster FcRs on the cell surface (7,8). Depending on the receptors engaged, the clustering of the extracellular domains triggers phosphorylation of tyrosine in the ITAMs or ITIMs, which subsequently recruits signaling molecules that promote a cellular response (9). The types of FcR-mediated effector cell responses are diverse and include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), release of cytokines and antigen uptake for presentation (10-14). FcRs are critical for maintaining immune system homeostasis as well as preventing pathogenic infections and they play a major role in inflammatory diseases and autoimmune disorders (9,13,15-17). The combination of distinct antagonistic and synergistic factors contribute to a considerable functional diversity within this group of antibody receptors. In this chapter we will discuss multiple factors which influence the antibody:FcR interaction and modify the immune response with a focus on the role of N-glycosylation (Figure 1.1).
Receptor presentation at the cell surface (JTR)
FcRs are predominately expressed on cells originating from hematopoietic progenitor stem cells including dendritic cells, neutrophils, basophils, eosinophils, macrophages, monocytes, mast cells, NK cells, B cells, a subset of T cells, and platelets as well as non-hematopoietic cell types such as syncytiotrophoblasts at various levels (18-20). FcR expression varies depending on cell lineage; not 4 surprisingly gene copy number is also implicated in disease. These factors can greatly influence the dynamic ability of the immune system to respond to the diverse repertoire of foreign invaders. Thus, variable surface expression by different immune cell types influences how the immune system responds to a foreign invader. This section will cover the cellular expression of FcRs and immune modulation of expression through downregulation and induction.
Five activating FcRs are expressed in humans. The highest affinity, CD64, is expressed on monocytes, dendritic cells and macrophages (11), mast cells (21), and neutrophils following IFN- exposure (22, 23). The low affinity CD32a is expressed on mast cells, neutrophils, macrophages, eosinophils and platelets (24). CD32c is expressed by 7-15% of individuals on NK cells and B cells and results from a gene mutation (4). The high/moderate affinity CD16a is expressed predominantly on NK cells, a subset of monocytes, mast cells, basophils, macrophages and is inducible in CD4+ T- cells (25, 26). The low/moderate affinity CD16b is found only in humans and expressed predominantly on neutrophils (27), a subset of basophils (28) and has inducible expression on eosinophils (29, 30). CD32b is the sole inhibitory receptor and is expressed on basophils, B cells, macrophages, dendritic cells, a subset of monocytes and neutrophils (24). Interestingly, CD32b is also expressed in non-hematopoeitic cells, including the endothelium of various organs (31).
Variability in receptor amount (JTR)
Gene duplications in individuals lead to copy number variation (CNV) of some
FcRs in the population. Surprisingly, only CD16a, CD16b and CD32c of the FcRs 5 exhibited CNV in a sample population of 600 subjects (32). CNVs have been correlated to autoimmune disorders as well as variations in surface expression levels. CNV of
CD16b is correlated to surface expression on neutrophils and implicated in SLE susceptibility (33, 34), as well as other autoimmune disorders (35, 36). Furthermore,
CD16a CNV appears to be functionally significant since increased surface expression positively correlated with increasing CD16a gene number (ranging from one to three copies) (32, 35). A CD16a indel has been shown to increase surface expression as well
(37).
FcR amount at the cell surface varies by cell type and receptor identity. On neutrophils, there are an estimated 100,000-300,000 surface exposed CD16b molecules and 10,000-40,000 CD32a molecules (38, 39). The predominant monocyte subtype at roughly 80% of the pool, “classical” monocytes, does not express CD16a.
“Non-classical” monocytes express CD16a at a level of roughly 10,000 CD16a molecules per cell but upon differentiation into macrophages express 40,000 CD16a molecules per cell while CD32 remained the same at approximately 10,000 molecules per cell (40). Another study found macrophages express 5-10 fold higher CD64, CD32a and CD32b while CD16a expression was comparable to nonclassical monocytes. M2c macrophages expressed overall higher levels of FcRs than M1 macrophages with the following order of expression: CD32a, CD32b > CD64 > CD16a (41). A high number of
CD16a molecules are expressed on CD16+ NK cells (100,000-250,000) (42).
Expression levels also vary based on the cell status. Following activation, innate immune cells can induce expression of FcRs (23, 25, 29, 30, 35). There is also evidence of receptor downregulation upon activation. Downregulation mechanisms 6 include both decreases in expression as well as shedding FcR from the cell surface following metalloproteinase cleavage. CD32a is shed from Langerhans cells and also expressed as a soluble form (43). CD32b is shed upon activation of B-cells (44). CD16a and CD16b are likewise shed upon activation of NK cells and neutrophils at a known cleavage site by the metalloprotease ADAM17 (45-48). Intriguingly, sCD16b is relatively abundant in serum (~5 nM) (49) and levels vary based on the immune state of the individual (50). Surprisingly, CD64 is the only human FcγR in which a soluble, serum- borne form has not been reported. This may be explained by the presence of a third extracellular CD64 domain in place of the cleavage site found in CD32s and CD16s.
Soluble FcR forms modulate immune responses. Soluble CD16b binds myeloid cells, NK cells, subsets of T cells, B cells and monocytes through complement receptor
3 (CR3 or Mac-1 or M 2, comprised of CD11b/CD18) and complement receptor 4
(CR4 or x 2, co mprised of CD11c/CD18). These interactions cause the release of
IL6 and IL8 by monocytes and indicate a potential role for soluble CD16b in inflammation (51). Shedding of CD16a from NK cells allows disengagement of the immune synapse from the target cell and the subsequent ability to kill again. One study demonstrated that repeated engagement by CD16a depleted perforin, however, shedding of CD16a allowed perforin replenishment upon subsequent activation by another activating receptor, NK group member D (NKG2D), which recognizes ligands not normally expressed on healthy tissue (52). Thus, it appears that the act of shedding of CD16 can allow disengagement of the foreign particle which would be crucial for the immune cell’s survival and preservation of potential future cytolytic activity. Though shed receptors are proinflammatory and recruit immune cells as discussed above, a 7 complete picture of the mechanisms of regulating surface expression upon immune activation is not currently available.
Receptor clustering at the membrane is required for effector function (JTR)
The correct presentation of FcRs on the cell membrane is essential for proper immune cell function. ADCC can destroy virally infected cells and cancer cells, and is thus a target for monoclonal antibody (mAb) therapies (53). ADCP is also an important mechanism in mAb therapy targeting malignant cells (14). ADCC and ADCP are dependent on the ability of low to moderate affinity FcRs to cluster on fluid plasma membranes for activation to occur (54) (Figure 1.1). Equally important is the regulation of these receptors when no activation signal is present.
Proper activation of FcRs following Fc engagement by macrophages requires clustering of FcRs and the displacement of inhibitory receptors. In one study utilizing murine RAW 264.7 cells, segregation of CD45, a phosphatase responsible for dephosphorylating ITAMs, is dependent on antigen distance from the target membrane
(55) (Figure 1.1). It appears that if the antibody is >10 nm from the target surface, there is a substantially impaired ADCP response. This phenomenon is due to the location of the epitope; epitopes closer to the surface exclude the inhibitory CD45 molecule (which stands ~22 nm tall vs. FcR-IgG complex = 11.5 nm) from the immune synapse.
Interestingly, a follow-up study that focused on FDA-approved mAbs found the targets were small surface proteins (less than 10 nm in height) suggesting there may be a requirement for mAb epitopes to be located close to the surface for therapeutic efficacy.
CD45 was also excluded from the immune synapse in activated human T cells (56). 8
Another study concerning inhibitory module segregation on human macrophages demonstrates CD64, but not CD32a, and inhibitory signal regulating protein (SIRP), in conjunction with CD47 (a receptor that inhibits macrophage phagocytosis), are clustered on quiescent cells but upon activation segregate in a process regulated by spleen tyrosine kinase (SYK)-dependent actin cytoskeleton reorganization (57).
Recently, FcR diffusion has been shown to be inhibited by the CD44 transmembrane protein which is immobilized by linearized actin filaments via ezrin/radixen/moesin
(ERM) and binds hyaluronan in the glycocalyx (58). This study used primary human macrophages as well as murine cell lines and murine models, utilizing single particle tracking and found CD44 and hyaluronan decreased the diffusion rate of FcRs, while also sterically blocking the binding of FcRs to immune complexes (Figure 1.1).
Receptor clustering overwhelms constitutive inhibition as described previously, allowing phosphorylation of the ITAM. ITAMs are phosphorylated via SYK, Src family kinase (SFKs) or -chain-associated protein kinase 70 (ZAP-70) for downstream activation of phosphoinositide-3-kinase (PI3K), NF-B, extracellular signal regulated kinase (ERK), phosphatidyl inositol 4-phosphate 5-kinase (PIP5K) , GTPases and other SRC-family kinases (53,54, 59, 60). Along with FcR clustering, actin polymerization and depolymerization is equally important for phagocytosis in RAW
264.7 macrophages by creating lammellipodium/pseudopods. These protrusions are controlled by Rac GTPase and lipid composition (54, 59) (Figure 1.1). Clustering has also been observed on the plasma membrane of murine derived macrophages using total internal reflection microscopy (TIRF) of a lipid bilayer supporting IgG (61). The
FcR microcluster appears on the macrophage pseudopod edge and is subsequently 9 transported to a synapse-like structure thereby recruiting SYK and production of
PtdIns(3,4,5)P3 coordinated with lamellar actin polymerization. Another study on quiescent human macrophages found lateral diffusion of FcRs is regulated by tonic activity of SYK causing actin cytoskeleton organization to increase the likelihood of
FcRs to be pre-clustered upon finding a pathogen (62).This study further described differential FcR mobility upon activation. FcRs at the periphery of the actin-rich pseudopod were more mobile than those already immobilized by binding of IgG-rich regions. The authors explained that this mobility difference is controlled by SYK- mediated regulation of the actin-cytoskeleton which would increase the likelihood of
FcRs to engage more IgG molecules at the leading edge of the lamellipodium/pseudopod and not waste time diffusing into already IgG-dependent,
FcR-immobilized, actin-rich rich regions of plasma membrane. Mobility of FcRs was described earlier to be decreased at the trailing end of polarized macrophages by CD44 that was bound to linear actin and connected to hyaluronan (58). It was also found in this study that on the leading edge of polarized macrophages, the side that encounters opsonized material, Arp2/3-driven actin branching predominates, initiated by phosphotidlyinosotide (3,4,5)-trisphosphate production, and increased FcR mobility allowing for more efficient clustering at the immune synapse. When Arp2/3-driven actin branching predominates, it was found CD44 is more mobile allowing greater FcR mobility (Figure 1.1).
In the human NK92 cell line, transduced to express CD16, a study showed 2 integrins mediate the dynamics of FcR receptor microclusters in a protein-tyrosine kinase 2 (Pyk2)-dependent manner, controlling the rate of target cell destruction by 10
ADCC (63). 2 integrins bind ICAM-1 on the target cell allowing adhesion and signal transduction through Pyk2 for actin remodeling and the subsequent enhancement of
FcR mobility. Furthermore, sites of granule release are surrounded by clusters of CD16 and release points are devoid of actin. Human NK cell lytic granules also converge at the surface in a dynein and integrin-signal dependent manner which aids spatial targeting of the weaponized molecules to limit off-target damage (64). Surprisingly,
CD16a is essential for ADCC of human CD16+ monocytes and upon CD16a engagement, 2 integrins are activated along with TNF secretion thereby indicating that non-classical monocytes (CD16+) are the sole monocyte class capable of ADCC
(65).
During the early stages of phagocytosis by RAW 264.7 cells, direct contact between FcRs and IgG is increased by greater IgG density on particles, and increased
IgG density results in an increased level of early signals. However, late stage signals are “all or nothing,” not concentration dependent, and regulated by PI3K concentration in the phagocytic membranes (66). In this study, low IgG density decreased the amount of opsonized particles but not the rate of phagosome formation and low IgG density particles that did result in phagocytosis recruited the same amounts of late stage signaling molecules (PIP3, Protein kinase C ε type, p85 subunit) and actin. Overall it appears that FcRs control the initial binding process essential for scanning the foreign particle and initial activation by binding IgG and later stages of commitment to destruction of the particle are controlled by both IgG density and membrane lipid composition. 11
On murine and human macrophages, receptor clustering upon activation is consistent with a change in the heterogeneity of the membrane lipid composition to a highly ordered phagosomal membrane that is heavily enriched in sphingolipids and ceramide but lacking cholesterol (67) (Figure 1.1). The authors state that lipid remodeling mediates F-actin remodeling and the biophysical characteristics of the phagosomal membrane are essential for phagocytosis. On human B cells, a polymorphism of the inhibitory receptor CD32b (Ile232Thr) located in the middle of the transmembrane domain, is described to decrease inhibitory function (68). This mutation was shown to result in aberrant localization to a sphingolipid and cholesterol rich region in contrast to the Ile232 wild-type. Aberrant localization is not surprising considering the introduction of a polar residue into the transmembrane domain (69). Furthermore, the ability of CD32b to inhibit B cell receptor (BCR)-mediated PIP3 production, AKT, phospholipase C--2 (PLC2) activation and calcium mobilization was impaired in cells expressing the CD16b Thr232 allotype as compared to Ile232. The authors indicate the
FcR locus was associated with SLE and this polymorphism may promote disease.
Thus, it appears lipid composition is important for FcR-mediated mechanisms.
The unique construction of CD16b indicates the potential for a different activation mechanism for neutrophils. Neutrophils predominantly express CD16b with 10-fold less
CD32a. CD32a signal transduction is well described and thought to be the canonical
FcR signal transduction via phosphorylation of ITAMs and subsequent SYK recruitment
(70). However, CD16b contains a GPI anchor and does not have a polypeptide transmembrane domain nor is it known to associate with a signaling coreceptor, therefore, it is unclear how CD16b promotes signaling in neutrophils. CD16b plays a 12 role in the initial binding of immune complexes in concert with 2 integrins (71).
Currently there are conflicting studies suggesting that CD16b can transduce a signal on its own (70, 73, 73), or it transduces a signal by acting with CD32a (74). A recent study found that CD16b cross-linking increased IL-10 and TNF expression, phosphorylated
SHP-2 in a lipid-raft mediated manner and inhibited apoptosis in neutrophils. Lipid composition certainly may be an important part of CD16b signal transduction in mechanisms similar to those discussed previously for macrophage phagocytosis and
CD32b on B-cells, however the role of lipids in neutrophil activation is not understood
(75-81). Interestingly proteinase 3 (PR3), CD16b, cytochrome b558, and NADPH oxidase co-immunoprecipitate on lipid rafts and PR3 and CD16b colocalize in confocal imaging suggesting these may interact in a lipid raft (75). Other findings suggest CD16b signals in conjunction with CR3 via lectin-like interactions (82), leading to neutrophil respiratory bursts (72). The function of GPI-linked CD16b remains undefined despite the high abundance of CD16b in the body and critical roles in mAb therapies (83).
The type of FcR membrane anchor impacts activation (JTR)
There are clear differences between the signaling and antibody-binding affinity of soluble and membrane-anchored FcR forms. However, less is known about the effects of the specific FcR membrane anchors on affinity and cell activation. All FcRs are localized to the membrane by a transmembrane polypeptide moiety or a glycosylphosphatidylinositol (GPI) moiety (CD16b only). A micropipette adhesion assay demonstrated that CD16a attached to microspheres via a GPI anchor bound roughly 5- fold tighter to IgG1-coated red blood cells (RBCs) than CD16a tethered by a 13 transmembrane domain (84, 85). Interestingly, it also appears that IgG1-coated spheres treated with phosphoinositide phospholipase C (PIPLC) to remove the diacylglycerol moiety bound to GPI-linked CD16a with 12-fold less affinity. These authors observed a
60-fold decrease when the GPI-anchor was completely removed. A CD16b-GPI construct showed 2-fold decrease of affinity upon PIPLC treatment and an 11-fold decrease following removal of the GPI-anchor. The authors hypothesized that enhancement of binding affinity associated with the GPI anchor may be due to an allosteric effect on CD16, changing the structure to bind IgG more effectively; such an allosteric mechanism was observed with other GPI-anchored proteins (80). Further studies will be required to fully elucidate how the GPI-anchor affects CD16b and how specific aspects of the membrane anchor confers distinct properties in vivo.
N-linked glycosylation heterogeneity depends on multiple factors (JTR)
Nearly half of all human proteins are estimated to be N-glycosylated making it one of the most abundant post-translational modifications (PTMs) (86). When a polypeptide contains the sequon N-X-S/T (X not being proline), a 14-residue sugar attached to the membrane through a dolichol phosphate is transferred by oligosaccharryl transferase (OST) en bloc to the nascent polypeptide asparagine co- tranlsationally (Figure 1.2) (87). N-glycosylation on the newly synthesized protein then serves functions in protein-folding and quality control pathways. Additionally, N-glycans protect the protein from proteolysis, reduce antigenicity, extend half-life in serum, promote secretion, contribute epitopes for receptor binding and affect protein structure
(88-89). Glycosidases trim the N-glycans on properly folded glycoproteins upon 14 transportation through the ER and golgi network while glycosyl transferases transfer specific monosaccharides to the N-glycan. This processing can result in vast differences in N-glycan structure which lead to a heterogenous population of N-glycans in vivo.
There are three main types of N-glycans; oligomannose, hybrid and complex-type
(Figure 1.2). Oligomannose-type N-glycans are ‘minimally’ processed which escape remodeling by glycosyl transferases while being trafficked through the golgi network to the membrane surface or extracellular space. Complex-type N-glycans are highly processed by glycosyl transferases leading to complex, highly branched N-glycans.
Hybrid-type N-glycans experience intermediate processing; the 6 branch contains only mannose residues while the 3 branch can be highly decorated like the complex-type
N-glycan. N-glycosylation variation depends on enzyme abundance and localization along with substrate availability and abundance (90-91). Thus, N-glycosylation composition can be modulated by environmental factors as well as cell-type specific gene expression. 15
Figure 1.2. (JTR) N-glycosylation processing occurs in the ER and Golgi Network. A 14-sugar residue attached to the ER membrane through a dolichol phosphate is transferred by oligosaccharyl transferase co-translationally to a peptide containing the sequon, “N-X-S/T,” in which X is any amino acid residue except for proline. Glycosidases trim down the N-glycan as it is passed through the ER and cis Golgi Network. Gnt1 transfers an N-acetyl glucosamine to the 3 arm of the Man5 N-glycan allowing processing by downstream glycosyl transferases in the medial and trans Golgi. Enzymes in the medial and cis Golgi can modify the N-glycan with various monosaccharides as well as add branches allowing a diverse repertoire of N-glycan structure possibilities. Glycoproteins are then secreted or moved to the plasma membrane through vesicle transport systems. The three main N-glycan types include oligomannose, complex and hybrid-types. Minimally processed, oligomannose-type N-glycans can sometimes escape processing and be released to the extracellular space or transported to the plasma membrane. Complex-type are highly processed N-glycans which can be highly modified and/or branched with N-acetyl neuraminic acid ‘caps’. Hybrid types contain only mannose monosaccharides on the arm like oliogmannose-type while they contain highly decorated modification on the 3 arm like complex-type N-glycans. 16
N-linked glycosylation of antibody affects FcRIII (CD16) binding (JTR)
IgG1 antibody affinities to FcRs are tuned by N-glycan composition as well as
FcR polymorphisms. The CD16a V158 allele increases affinity 3-5 fold as compared to the CD16 F158 allele (92). mAb therapies have been described to have increased clinical efficacy in homozygous individuals with the higher affinity CD16a V158 allele as compared to homozygotes with the lower affinity CD16a F158 allele, directly correlating affinity to enhanced clinical outcomes (93-94). The N-glycan composition on IgG1 has been well described to modulate affinity to FcRIIIa (CD16a) and FcRIIIb (CD16b) (95-
102). The removal of fucose enhances affinity while addition of galactose increases affinity. Furthermore, a second generation of glycoengineered mAbs designed to take advantage of this effect have been described to enhance treatment outcomes, most likely due to these affinity enhancements (95-99). In addition to glycoengineered mAb therapies, serum IgG N-glycosylation has also become of interest because N-glycan composition changes with disease state as well as gender, infection, pregnancy and age which has been recently extensively reviewed by Gudelj et. al., 2018. (103-107).
Previous studies have mainly focused on IgG glycosylation as compared to CD16a N- glycosylation most likely because of the shear abundance of IgG in serum but have not looked at CD16a N-glycosylation.
CD16a N-glycosylation composition affects binding to IgG1 (JTR)
CD16a is N-glycosylated at 5 sites; N38, N45, N74, N162 and N169, and is expressed predominantly on NK cells and nonclassical monocytes as described in previous sections. CD16a N-glycosylation is important in its interaction with IgG. Edberg 17 and Kimberly found CD16a from NK cells expressed high amounts of oligomannose- type N-glycans using lectin studies (108). Recent higher resolution mass spectrometry analysis of recombinant sources of CD16a N-glycosyation have been described to be much different to these lectin binding studies, granted these systems are expected to glycosylate proteins differently (101-102, 109-110). Edberg and Kimberly also found
CD16 from NK cells bound IgG tighter than CD16 from monocytes and neutrophils suggesting differences in post-translational modifications to be the cause of this discrepancy (108). As described in previous sections, neutrophils express CD16b, a lower affinity FcR as compared to CD16a thus it was to be expected CD16 from neutrophils would bind IgG weaker (111-112). The reason for the vast differences in affinity between the two very similar proteins, CD16a and CD16b is detailed in Chapter
3. sCD16b N-glycosylation from serum has recently been described by two groups and indeed express different glycoforms as compared to recombinant sources (113-114).
Interestingly, these studies of CD16b from serum found N45 to express predominantly oligomannose-type N-glycans suggesting restricted processing at this site.
CD16a N-glycan composition tunes affinity to IgG1. As described in a previous section IgG glycosylation is known to be able to affect protein interactions. Hayes et. al,
2017 described differential binding kinetics of CD16a glycoforms expressed in NSO,
CHO and HEK293f cells (102). Our group recently characterized the role of oligomannose-type N-glycosylation on CD16a as compared to complex-type (115). This study found CD16a is the only FcR which gets a significant enhancement in binding affinity (10-50-fold) when displaying an oligomannose-type N-glycan as compared to a complex-type, and this effect is specific to the N162 N-glycan. Previous studies by 18 independent groups have implicated the IgG fucose to prevent stabilizing carbohydrate- carbohydrate interactions between the N162 N-glycan and the IgG1 N297 N-glycan
(116-117). However, Falconer et. al. in our group thoroughly characterized the role of the N162 N-glycan using ITC, SPR and MD and found the N162 glycan clashes with the
IgG1 N-glycan showing there was no stabilizing role of the N162 N-glycan (118). Thus,
N-glycan composition on CD16a an N162 could tune affinity to antibody in vivo.
The discovery that CD16a N-glycan composition affects binding to IgG prompted our lab to develop methods to characterize N-glycosylation of CD16a from primary tissue. This has most likely not been performed before because material is extremely limited and technological advances in mass spectrometry in recent years have reached the sensitivity levels needed for the analysis. We estimate 1.5 liters of fresh blood would be needed to isolate enough cells to thoroughly characterize CD16a from NK cells and perhaps more from monocytes. In Chapter 2 we also elucidate the reason for the stark affinity difference between the very similar FcR’s; CD16a and CD16b.
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CHAPTER 2. A SINGLE AMINO ACID DISTORTS THE FC RECEPTOR IIIB / CD16B STRUCTURE UPON BINDING IMMUNOGLOBULIN G1 AND REDUCES AFFINITY RELATIVE TO CD16A
A manuscript published in the Journal of Biological Chemistry.
Roberts, J. T. & Barb, A. W. A single amino acid distorts the Fc γ receptor IIIb / CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a. J. Biol. Chem. jbc.RA118.005273 (2018). doi:10.1074/jbc.RA118.005273
Abstract
Therapeutic mAbs engage Fc receptor III (CD16) to elicit a protective cell- mediated response and destroy the target tissue. Newer drugs designed to bind CD16a with increased affinity surprisingly also elicit protective CD16b-mediated responses.
However, it is unclear why immunoglobulin G (IgG) binds CD16a with more than 10-fold higher affinity than CD16b even though these receptors share > 97% identity. Here, we identified one residue, Gly129, that contributes to the greater IgG-binding affinity of
CD16a. The CD16b variant D129G bound IgG1 Fc with two-fold higher affinity than
CD16a and with 90-fold higher affinity than WT. Conversely, the binding affinity of
CD16a-G129D was decreased 128-fold relative to WT CD16a and comparably to that of
WT CD16b. The interaction of IgG1 Fc with CD16a, but not with CD16b, is known to be sensitive to the composition of the asparagine-linked carbohydrates (N-glycans) attached to the receptor. CD16a and CD16b-D129G displaying minimally processed oligomannose N-glycans bound to IgG1 Fc with about 5.2-fold increased affinity compared with variants with highly processed complex-type N-glycans. CD16b and the
CD16a-G129D variant exhibited a smaller 1.9-fold affinity increase with oligomannose 30
N-glycans. A model of glycosylated CD16b bound to IgG1 Fc determined to 2.2 Å resolution combined with a 250-ns all-atom molecular dynamics simulation showed that the larger Asp129 residue deformed the Fc-binding surface. These results reveal how
Asp-129 in CD16b affects its binding affinity for IgG1 Fc and suggest that antibodies engineered to engage CD16b with high affinity must accommodate the Asp-129 sidechain.
Introduction
Immunoglobulin G (IgG) opsonizes a previously encountered pathogen to target it for destruction by eliciting an immune response. IgG binds the target with high specificity through the antigen binding fragments (Fabs) and interacts with innate immune system effector cells through the crystallizable fragment (Fc) by engaging Fc receptors (FcRs), thus bridging the adaptive and innate immune systems (Figure 2.1).
There are three classes of cognate human FcRs: FcRI (CD64), FcRII (CD32) and
FcRIII (CD16) (1). CD16 is of particular interest because it is the primary receptor responsible for antibody-dependent cell mediated cytotoxicity (ADCC) and thus tumor clearance (2,3). Two CD16 forms, CD16a and CD16b, bind to IgG1 with surprisingly different affinities considering the high degree of structural and sequence similarity
(4,5). In this manuscript we probe the contribution of an amino acid residue to the different binding affinities of CD16a and CD16b.
CD16a is an integral membrane protein predominantly expressed on NK cells and monocytes. CD16b is predominantly expressed with a glycosylphosphatidylinositol anchor on neutrophils, a subset of basophils and shows inducible expression on 31 eosinophils (6-12) (Figure 2.1). CD16a has two predominant alleles (V158 or F158) and
CD16b has two predominant alleles including human neutrophil antigen 1 (HNA-1 or
NA1) and NA2 (13,14). CD16a-V158 and CD16b-NA2 share >97% sequence identity and differ by only four amino acid residues in the extracellular antibody-binding domains
(Figure 2.1B). Despite the significant homology shared by CD16a and CD16b, CD16a binds antibody more than 10-fold tighter though it is unclear how differences in these four amino acids contribute to differences in binding affinity (5). Only one of these four differences, at position 129, forms part of the IgG1-binding interface.
FcRs are heavily modified with asparagine-linked carbohydrates (N-glycans; for a review of N-glycosylation see (15)). CD16a has as many as five N-glycans and
CD16b (NA2) has as many as six though it is not clear if all sites are fully occupied. N- glycan composition of CD16 appears to be varied, depending upon the type of expressing cell and it was shown that different cells bind IgG with different affinity through CD16 (16-19). Three recent studies reported that the binding affinity of CD16a is influenced by receptor N-glycan composition (19-21). In one example, CD16a with minimally-processed Man5 N-glycans bound 12-fold tighter to IgG1 Fc (G0F) than
CD16a with highly-processed and elaborated complex-type N-glycans but glycan composition only impacted CD16b affinity by less than 2-fold (examples of different N- glycoforms are shown in Figure 2.2) (19,22). This result is surprising considering the significant homology shared by CD16a and CD16b (Figure 2.1). The CD16a N-glycan at position 162 mediated this effect that likewise may be influenced by the nearby Gly-
129 residue (20). 32
Figure 2.1. Fc receptor (FcR) III/CD16 is expressed in two forms from two distinct genes. A) FcRIIIb/CD16b is highly expressed on neutrophils and FcRIIIa/CD16a is the predominant FcR expressed on natural killer (NK) cells. B) These proteins differ by only four amino acid residues (highlighted in yellow) in the mature proteins. The N-terminal signal peptide is indicated in red and is not found in the fully processed proteins. The C-terminal residues of CD16b shown in orange are cleaved prior to addition of the GPI anchor at the newly uncovered C-terminal serine. N-glycosylation sites are indicated with grey shading. Both proteins are cleaved by ADAM17 and released from the cell surface following cell activation. Resolved N-glycan residues are shown as spheres.
33
N-glycan composition of the IgG1 crystallizable fragment (Fc) is a well-known variable in CD16 binding affinity (3,23-26). This property directly impacts the development of therapeutic monoclonal antibodies (mAbs) because increasing affinity for CD16 increased therapeutic efficacy (13,25,27-29). A major advance for mAb development occurred with the observation that IgG lacking a prevalent antibody modification, L-fucose attached to the core (1)GlcNAc residue of the N-glycan, increased binding affinity and ADCC (30). This observation inspired the development of many new glycoengineered mAb therapies (31-34). Notably, CD16b demonstrated a larger enhancement in binding non-fucosylated IgG1 Fc in vitro when compared to
CD16a in vitro (5). The current generation of glycoengineered antibodies are more effective at binding to CD16b on neutrophils and eliciting effector responses, showing greater therapeutic potential (35,36). These studies support the development of mAbs that bind CD16b with higher affinity to mobilize an effective neutrophil response.
However, the development of future mAbs is limited by a lack of information regarding the detailed mechanism and identification of which residues contribute to the reduced affinity of CD16b for IgG1 Fc when compared to CD16a.
A comparison of the amino acid sequences for CD16b and CD16a reveals only one of the four differences in the antibody-binding domains, at position 129, directly contributes the interface formed with IgG1 Fc (Figure 2.1B). If Asp-129 of CD16b disrupts this interface, then CD16b D129G is expected to bind as tightly as CD16a and
CD16a G129D will bind with less affinity. Furthermore, if CD16a Gly-129 contributes to the sensitivity of affinity to receptor N-glycan composition, then CD16a G129D should no longer be sensitive to N-glycan composition. In this manuscript, we evaluate how 34
Gly-129 and Asp-129 impact the affinity of CD16 for IgG1 Fc. We also determined how these residues modulate the differential sensitivity of CD16a and CD16b to N-glycan composition.
Figure 2.2. Total ion current of procainamide-derivatized CD16b N-glycans expressed in two distinct glycoforms and analyzed by HILIC-MS. (top) CD16b was expressed to display oligomannose (Man5) N- glycans. (bottom) CD16b with complex-type (CT) N-glycans.
Results
N-glycomics of the recombinant proteins
In order to evaluate the impact of N-glycan composition and Asp-129 v. Gly-129 on CD16 structure and IgG1 Fc binding, we expressed four CD16 variants including
CD16a, CD16a G129D, CD16b and CD16b D129G, using two cells lines that resulted in a panel of eight receptor variants. One cell line, HEK293F, contains a large repertoire of glycan modifying enzymes and expressed CD16 with a heterogeneous mixture of highly branched complex-type N-glycans (Figure 2.2). The second cell line, HEK293S, contains a defect in the GNT1 gene (37,38) and expressed CD16 with predominantly 35
Man5 oligomannose-type N-glycans though small amounts of Man6, Man7, Man8 and
Man9 were also present (Figure 2.2).
rCD16b D129G-CT binds IgG1 Fc with affinity comparable to rCD16a-CT
Wild type recombinant (r)CD16a-CT bound IgG1 Fc glycoforms with affinities between 38- and 60-fold tighter than rCD16b (Figure 2.3 and Table 2.1). These values were comparable to previous results from our laboratory, however, the binding of rCD16a-CT is roughly 1.5 to 4-fold tighter (5,20). As predicted, rCD16a-CT bound IgG1
Fc with more than 100-fold greater affinity than rCD16a G129D-CT. The rCD16a
G129D-CT variant bound with an affinity comparable to rCD16b-CT (22 mM v 6.4 mM, respectively; Figure 2.4). In the complementary experiment, rCD16b-D129G-CT bound at least 64-fold tighter to three IgG1 Fc N-glycoforms than rCD16b-CT. Furthermore, the affinities of rCD16a-CT and rCD16b D129G-CT were less than 3.1-fold different, confirming the importance of a Gly at position 129 in high-affinity IgG1 Fc binding. It is possible that subtle differences in N-glycan composition resulting from expression conditions, in particular the N-glycan at Asn-162, impact the measured affinity. This effect is noted for CD16b D129G-CT that contains a larger amount of oligomannose N- glycans compared to CD16a (Figure 2.2).
36
Figure 2.4. A glycine at position 129 of CD16 is essential for high affinity IgG1 Fc binding. Dissociation constants indicate the impact of the IgG1 Fc N-glycan, CD16 in N-glycan and residue at position 129 on binding affinity. Error bars indicate the error-of-fit for the interactions.
Table 2.1. Binding affinity measurements with two receptor glycoforms and three IgG1 Fc glycoforms determined by surface plasmon resonance. IgG1 Fc G0F IgG1 Fc G2F IgG1 Fc G0
Receptor KD (nM) ± err KD (nM) ± err KD (nM) ± err rCD16a-CT 140 10 70 2 25 1 rCD16a G129D-CT 22000 1000 12500 600 3000 200 rCD16b-CT 6400 400 4200 300 950 100 rCD16b D129G-CT 100 10 48 3 8 1 rCD16a-Man5 48 2 22 1 3.9* 1.0 rCD16a G129D-Man5 9500 300 4000 200 1100 100 rCD16b-Man5 5200 300 2900 200 560 30 rCD16b D129G-Man5 13 1 6.0 0.3 3.6* 1.0 rCD16b N38Q/N64Q/N74Q/N169Q-Man5 6400 600 4000 400 650 50
*- KD calculated from kinetic data
37
Tighter-binding CD16 variants are sensitive to N-glycan composition
Comparing the binding affinities of the CD16 variants with different N-glycan composition provided insight into the role of Gly-129 in the higher affinity interactions.
All receptor variants bound tighter with oligomannose N-glycans, however, receptors with a Gly residue at position 129 showed an average affinity increase of 5.2 ±2.6-fold compared to 1.9 ±0.8-fold for receptors with an Asp residue at position 129 (Table 2.1).
The CD16 129 residue impacts association and dissociation of IgG1 Fc
The receptor variants that bound with lower affinity revealed very fast apparent association and dissociation kinetics using the relatively high concentrations of analytes required to reach saturation (CD16b and CD16a G129D). These rates were too rapid to accurately estimate from the SPR sensorgrams because binding was limited by the rate of analyte flow over the chip surface (Figure 2.3). Conversely, variants that bound with higher affinity showed slower observed association and dissociation rates (CD16a and
CD16b D129G). These observed rates appeared slow due to the small amounts of receptor used for the binding measurements and are not comparable to the association rates measured for the low affinity variants that were tested at much higher analyte concentrations and likely bind with equal or slower on rates. As a result, the associations for the high affinity variants failed to reach equilibrium after 800 s and only an analysis of binding kinetics provided appropriate estimates of KD (Figure 2.3 and
Table 2.2). Reactions that permitted KD measurement using both equilibrium and kinetic data revealed comparable results and validated the direct comparison of KDs measured from both types of data (Tables 2.1&2.2). 38
Figure 2.3. The binding affinity and binding kinetics of the CD16b D129G protein are comparable to the high affinity CD16a protein. Shown are representative binding sensorgrams for the CD16 variants binding to IgG1 Fc (G0 glycoform). Dissociation constants are determined using equilibrium intensity values with the exception of those KDs marked with an asterisk (*) that were calculated by fitting kinetic data. Errors indicate uncertainties from the curve fitting procedures. 39
Table 2.2. Association and dissociation rate constants measured from kinetic fits of the surface plasmon resonance sensorgrams. IgG1 Fc G0F IgG1 Fc G0
kON ± err kOFF ± err KD kON ± err kOFF ± err KD (nM) (nM) Receptor (mM-1s-1) (s-1) (mM-1s-1) (s-1) rCD16a-CT 39 11% 0.00506 14% 130 98 13% 0.00420 10% 43 rCD16b D129G-CT 306 18% 0.01853 16% 61 317 4% 0.00323 10% 100 rCD16a-Man5 437 19% 0.01729 16% 40 625 1% 0.00242 29% 3.9 rCD16b D129G-Man5 422 2% 0.00440 18% 10 685 4% 0.00246 20% 3.6
Among the high-affinity variants, it is notable that rCD16a-CT bound to IgG1 Fc
G0F and G0 with the slowest of the measured on-rates (39 and 98 mM-1 s-1,
respectively, compared to 306–685 mM-1 s-1; Table 2.2). Surprisingly, rCD16b D129G
bound with a 3 to 8-fold faster kON compared to rCD16a but more similar kOFFs (0.8 to
3.7-fold difference). These differences contribute to KDs that were no more than 2.1 to
2.3-fold different. This result indicates that substitution at residue 129 does not transfer
the complete kinetic properties between CD16a and CD16b.
The structure of glycosylated CD16b in complex with IgG1 Fc
Three models of IgG1 Fc bound to non-glycosylated CD16b revealed the
structural features of low-affinity FcRs bound to antibody (39,40). However, the
availability of moderate resolution diffraction (3.0–3.5 Å), moderate Rfrees (0.28–0.36)
and high temperature factors for the Asp-129 backbone atoms (78-104 Å2) limit the
ability to formulate hypotheses regarding the position of individual atoms at the Fc-
receptor interface and compare to three higher quality structures of the CD16a:IgG1 Fc
complex (2.2 Å with Rfrees = 0.24–0.26 (20,41,42)). One feature of the higher-quality
CD16a complexes is the presence of the Asn-45 and Asn-162-glycans. Multiple reports 40 highlighted the importance of these two N-glycans and the dispensability of the remaining three N-glycans to IgG1 Fc binding affinity (20,43,44). To improve the crystal quality we prepared glycosylated rCD16b-Man5 with four of the six N-glycosylation sites removed by mutating the modified Asn residue to a Gln (N38Q/N64Q/N74Q/N169Q).
Indeed, this rCD16b-Man5 variant bound IgG1 Fc with 0.7 to 0.9-fold of the affinity observed using rCD16b-Man5 and indicated a suitability for crystallization trials (Table
2.1).
Figure 2.5. CD16a binds IgG1 Fc with greater affinity and makes a closer approach to IgG1 Fc through the loop containing residue 129. The overall complexes formed by A) CD16a and B) CD16b with IgG1 Fc are highly similar with an rmsd = 0.499 Å. Protein backbone is shown as ribbons and N-glycans are shown as sticks. C) Electron density of the CD16b region surrounding Asp-129 is well resolved at 1.5 . D) The Gly-129 amide nitrogen atom of CD16a is located 1.3 Å closer to the carbonyl carbon of the IgG1 Fc Asn-297 than E) the amide nitrogen of Asp-129 from CD16B. 41
Two crystal forms of the rCD16b-Man5:IgG1 Fc complex diffracted to high resolution. One form crystallized in the P212121 space group and diffracted to 2.5 Å
(data not shown). A second crystal in the C2 space group diffracted to 2.22 Å with high completeness (data not shown). The CD16a-IgG1 Fc complex provided initial phases
(20) and the iterative refinement of the higher resolution data resulted in an Rfree =
0.2477 as well as a model with no Ramachandran outliers and only 1.2% of sidechain rotamer outliers. As expected, the overall structure is highly similar to other CD16a and
CD16b models (Figure 2.5). This structural model has an r.m.s.d. of 0.499 Å when compared to the rCD16a-Man5:IgG1 Fc complex (pdb 5vu0; (20)) and 2.054 Å when compared to a non-glycosylated rCD16b:IgG1 Fc complex (pdb 1T89; (40)).
The Asp-129 residue of CD16b distorts the Fc-binding interface
In addition to higher resolution data and higher refinement scores when compared to previous CD16b:IgG1 Fc complexes, the model calculated from the 2.22 Å dataset provided low temperature factors for the interface residues with values for the
Asp-129 backbone atoms ranging from 42 to 43 Å2 (compared to 78-104 Å2 for previous
CD16b models). These lower values allow comparison to CD16a models with similar quality (Figure 2.5C). Despite the high degree of amino acid similarity, the interface formed between CD16b and IgG1 Fc is smaller and involves less residues than the interface formed by CD16a (data not shown). The buried surface area measured with this model is 860 Å2 and comparable to previous CD16b models (1t89= 737 Å2 and 42
1e4k = 816 Å2 (39,40)). These areas are less than those measured with the CD16a-
IgG1 Fc complex (3sgj = 1193 Å2, 3ay4 = 924 Å2, 5vu0 = 897 Å2 (20,41,45).
Figure 2.6. Asp-129 in CD16b decreases the distance between beta strands and distorts the interface formed between the Asn-162-glycan and Arg-155 when compared to Gly-129 and CD16a. A) The CD16a complex with glycosylated CD16a is shown in red (pdb 5vu0 (20)) and superimposed on the unliganded (“unlig.”) CD16b (pdb 1e4j (39)) shown in gray. Note that the position of the Asp-129 sidechain clashes with the IgG1 Fc C'E loop. B) Glycosylated CD16b in complex with IgG1 Fc is shown in orange and superimposed on the unliganded (“unlig.”) CD16b shown in gray. IgG1 Fc binding distorts the conformation surrounding Asp-129, shortening the distance from Asp-129 to Arg-155. C&E) Two views of CD16a with the polypeptide surface shown in grey and relevant residues as red sticks. D&F) Two poses of CD16b with the polypeptide surface shown in grey and relevant residues as orange sticks. This Asp- 129-mediated strand distortion perturbs the interface formed between the (1)GlcNAc residue of the CD16 Asn-162-glycan and Arg-155. 43
Asp-129 represents the only amino acid difference between CD16b and CD16a that is found at the interface formed with IgG1 Fc. The close approach of the CD16a
Gly-129 amide nitrogen to the IgG1 Fc Tyr-296 carbonyl (3.2 Å) is prevented by the
CD16b Asp-129 residue, increasing this internuclear distance to 4.5 Å and distorting the
CD16b backbone (Figure 2.5D-E). Surprisingly, it appears that this distortion is not present in the absence of IgG1 Fc; a model of unliganded CD16b reveals an identical backbone conformation to CD16a in the region of Asp-129 (Figure 2.6A). The observed conformation in the unliganded CD16b model places the Asp-129 sidechain within the van der Waals radii of the IgG1 Fc Asn-297 residue. Thus, CD16b must deform to accommodate IgG1 Fc, distorting the backbone. These changes at the Fc interface potentially explain why CD16b binds IgG1 Fc weaker than CD16a.
The backbone distortion due to Asp-129 impacts the local tertiary structure, deforming the beta sheet and reducing the distance across the sheet by 1.2 Å when compared to CD16a (as measured by the distance to Arg-155; Figure 2.6A-B). This change impacts the conformation of the Arg-155 sidechain which forms an interface with the Asn-162-glycan (1)GlcNAc residue in the CD16a complex. In the CD16b complex, the contacts between Arg-155 and the (1)GlcNAc residue are reduced as the
Arg-155 sidechain shifts away from the carbohydrate (Figure 2.6C-F). This impact on the intramolecular interface formed between the Asn-162 glycan (1)GlcNAc residue and the Arg-155 sidechain may explain why the affinity of IgG1 for CD16a, but not CD16b, is sensitive to the receptor N-glycan composition (Figure 2.4 and Table 2.1).
44
MD simulations of the CD16b-IgG1 Fc complex
Measurements of binding affinity pinpointed the residue at position 129 as a critical difference between CD16a and CD16b. X-ray crystallography highlighted differences in the interfaces formed by these receptors and IgG1 Fc. However, these data represent a single conformational snapshot and it remained unclear whether X-ray crystallography only captured a representative state. The N-glycosylated CD16b-IgG1
Fc complex served as a starting point for a 250 ns all-atom molecular dynamics simulation to compare to a recent 250 ns simulation of the CD16a-IgG1 Fc complex to assess the stability of the observed interactions on this timescale (20). Though this simulation timescale is insufficient to probe all potential conformations accessed by these proteins, it may prove informative of differences in local conformation, potentially governed by low energetic barriers that would be sampled in a relatively restricted simulation.
Distances separating the CD16 129 residue and the IgG1 Fc C'E loop showed values consistent with the models determined by X-ray crystallography: the CD16b Gly-
129 C atom was 4.6 Å from the Fc Asn-297 C compared to 3.9 Å for the CD16a complex (Figure 2.6). Though the average distances observed with MD are slightly larger (5.0 and 4.3 Å, respectively), the differences of 0.7 Å are identical (Figure 2.7).
These greater distances for CD16b separated the CD16a Gly-129 N and Fc Tyr-296 O atoms (ave = 4.8 Å) which participated in a H-bond in the CD16a simulations (ave = 3.0
Å). In the CD16b simulations and model determined by x-ray crystallography, this H- bond was replaced by an interaction with the CD16b Lys-128 N atom which sampled 45 distances within H-bond range in the CD16b simulation but not in the CD16a simulation
(Figure 2.7, bottom panels).
Figure 2.7. Internuclear distances observed in crystallography are maintained during 250 ns all-atom molecular dynamics simulations of the CD16 – IgG1 Fc complexes. Panels in the left column indicate a 10-frame average of each distance over the course of the simulation. The right panels show histograms reporting the number of frames that correspond to each distance. Vertical arrows indicate the distance measured from structures determined by X-ray crystallography (CD16a – IgG1 Fc G0 (pdb 5vu0 (20)); CD16b – IgG1 Fc G0 this manuscript). Distances correspond to those measured in Figures 2.5 and 2.6. “X129” refers to residue Asp-129 of CD16b and Gly-129 of CD16a.
46
Simulation likewise preserved the observed deformation of the CD16b beta sheet induced by Asp-129 that reduced the distance between the Arg-155 and 129 C atoms by 1.2 Å compared to CD16a (Figure 2.6A and 2.B). Though the difference in averages is somewhat smaller in the simulations at 0.8 Å, the same trend is observed until the very end of the CD16b simulation (Figure 2.7). Interestingly, CD16b drifts slightly away from IgG1 Fc in the final 30 ns of the simulation as indicated by the increasing distances between CD16b Asp-129 and Fc residues Asn-297 and Tyr-296. At the same time, the beta sheet deformation also disappears and the distance between the CD16b Arg-155 and Gly-129 C atoms matches the CD16a simulation (9.6 Å). This observation supports the conclusion that complex formation and contact with the IgG1 Fc C'E loop impinges upon the CD16b Asp-129 residue.
Discussion
The data presented here demonstrate that a single amino acid residue at position
129 accounts for the major differences in binding affinity, binding kinetics, and sensitivity to receptor N-glycan composition. The presence of a Gly at CD16 residue 129 supports high affinity IgG1 Fc binding but an Asp residue, as found in CD16b, reduces affinity.
These data are supported by binding measurements and the structural model of the N- glycosylated CD16b-IgG1 Fc complex determined by X-ray crystallography. Extensive all-atom MD simulations further supported differences in the CD16-IgG1 Fc models.
The deformation of CD16b due to Asp-129 upon complex formation with IgG1 Fc likely contributes an energetic penalty that prevents CD16b from binding as tightly as
CD16a. This is evident upon examining a model of unliganded CD16b that showed 47 indistinguishable backbone geometry compared to CD16a in complex with IgG1 Fc
(Figure 2.6). Furthermore, this deformation reduced the buried surface area formed in the complex. With these data alone it is not possible to quantify the impact of the deformation on IgG1 Fc binding affinity because it is not clear how Asp-129 impacts conformational sampling of unliganded CD16b. Robinson et al. recently noted evidence for sampled conformation differences between CD16a and CD16b and developed a
CD16a-selective affimer, AfG3, that binds between the two extracellular domains to a region with conserved amino acids in CD16a and CD16b (46). Though the authors believe the selectivity of AfG3 is largely due to H-bonds formed by CD16a Arg-18
(CD16b NA2 S18), it is clear that amino acid differences distant from the IgG1-binding surface of CD16 can impact conformational sampling.
It is surprising that CD16a is more sensitive to N-glycan composition than
CD16b, and that this behavior is influenced by the residue at position 129. As stated,
CD16a N-glycan composition affects affinity for binding IgG1 Fc (16,19,21). The data presented here further support the recent observation that CD16a is the only FcR sensitive to receptor N-glycan composition (Table 2.1 and (22)). Furthermore, the analysis of the CD16 amino acid variants binding to IgG1 Fc highlights the central role of CD16a Gly-129 in this unexpected behavior. The structure of glycosylated CD16b in complex with IgG1 Fc provides some insight into the mechanism underlying this phenomenon: Asp-129 deforms the CD16b beta sheet adjacent to the interface formed with IgG1 Fc and this distortion perturbs contacts between the Asn-162-glycan and Arg-
155 (Figure 2.6). Though it is unclear why CD16a with Man5 N-glycans binds tighter to
IgG1 Fc than CD16a with complex-type N-glycans, the ability to increase the sensitivity 48 of CD16b to N-glycan composition by the D129G substitution indicates residues surrounding Gly-129 are likely involved in this phenomenon.
CD16b has received much less attention than CD16a. Though engaging CD16a is essential for the therapeutic activity of most mAbs, CD16b has emerged as an important target (35). The recent realization that targeting CD16b may improve these drugs is partially due to the roughly 50-fold higher amount of CD16b in the human body compared to CD16a. We estimate that the average person contains more than 200 g of CD16b from neutrophils in circulation compared to just 4 g of CD16a from monocytes and NK cells (2,19,47). Perhaps because of this difference in abundance, mAbs that specifically target CD16b provide definite advantages. One glycoengineered anti-CD20 antibody, obinutuzumab, showed greater therapeutic efficacy than the standard anti-CD20 therapy rituximab due to higher affinity and an ability to activate neutrophils through CD16b but not CD32a (35). Another study also found glycoengineered nonfucosylated rituximab similarly enhanced neutrophil activation through CD16b engagement (36). These findings demonstrate that increasing the
CD16b binding affinity of mAbs provides better therapeutic responses and potentially lower doses in the clinic. Our finding that the Asp-129 substantially decreases rCD16b affinity for IgG1 Fc indicates that designing antibodies to accommodate the Asp-129 sidechain may improve CD16b binding affinity and contribute to improved therapeutic outcomes.
49
Experimental Procedures
Materials
All materials were purchased from Sigma-Aldrich unless otherwise noted.
Protein expression and purification
CD16a (V158) and CD16b (NA2) were previously cloned into the Not1 and
EcoR1 restriction sites of the pGen2 vector (5,48,49). CD16a and CD16b cloned into a pUC57 vector served as templates to generate genes encoding mutated receptors according to the QuikChange protocol (Agilent). Mutations were verified by DNA sequencing at the ISU DNA Facility. Verified mutant genes were subcloned into the pGen2 plasmid using the Not1 and EcoR1 restriction enzymes (49).
The expression and purification of CD16 with complex-type N-glycans was achieved by expressing from HEK293F cells and purified as previously described (5).
CD16 with oligomannose (Man5) N-glycans was expressed using the HEK293S cell line as previously described (19). Typical yields were ~0.2 mg/mL of final culture volume.
IgG1 Fc used for binding analyses and single IgG1 Fc glycoforms were prepared as previously described (5). Binding analyses for the previous 2016 report and the results reported in this current manuscript were collected within a few days of one another.
IgG1 Fc lacking a fucose residue was used for crystallography and was prepared by expressing protein in the presence of 250 M 2-fluoro-2-deoxy fucose as previously described (5). Glycomics analyses of PNGaseF released N-glycans using HILIC-ESI-
MS/MS was performed as described (19).
50
Crystallization of CD16b-IgG1 Fc complex
CD16b-N38Q/N64Q/N74Q/N169Q with Man5 N-glycans was transferred to a buffer containing 50 mM tris(hydroxymethyl)aminomethane, 100 mM sodium chloride and 0.5 mM ethylenediaminetetraacetic acid, pH 8.0. A 1:50 molar ratio (receptor:TEV-
GFP) was added and the samples incubated overnight in the dark at room temperature.
CD16 was separated from GFP by flowing over a Ni-NTA resin (Qiagen) and concentrating the flow through fractions containing CD16. CD16 was exchanged into a buffer containing 20 mM 3-(N-morpholino)-propanesulfonic acid, 100 mM sodium chloride, pH 7.2 with an Amicon Ultra centrifugal filter with a 10 kDa molecular weight cutoff (Merck Millipore). CD16 was then mixed with IgG1 Fc (-fucose) at a (1:1.5) molar ratio. Complex was separated from unbound IgG1 Fc by purification with an AKTA pure chromatography system equipped with a Superdex 200 gel filtration column preequilibrated with a buffer containing 20 mM 3-(N-morpholino)propanesulfonic acid,
100 mM sodium chloride, pH 7.2 (GE Healthcare Bio-Sciences). Fractions containing the CD16:Fc complex were identified by SDS-PAGE and concentrated to 12 mg/mL.
Crystals in the P212121 space group were obtained using the hanging drop method with a well solution of 50 mM 2-(N-morpholino)ethanesulfonic acid, 10% PEG3350 (w/v) and 50 mM sodium chloride, pH 6.0. Crystals in the C2 space group were obtained using the hanging drop method with a well solution of 50 mM 2-(N-morpholino)- ethanesulfonic acid, 8% PEG3350 (w/v) and 60 mM sodium chloride. All crystals were cryoprotected with 15% ethylene glycol diluted with well solution and flash frozen in liquid nitrogen. Diffraction data was collected at 1° increments for 200 frames with an exposure time of 0.5 seconds on beamline 23-ID-D at the Argonne National Laboratory 51
Advanced Photon Source (APS) with a Pilatus3 6M detector. The software HKL-3000 was used to index, scale and merge datasets. Initial phases were determined with molecular replacements in Phenix-Phaser MR using the PDB id# 5vu0 (20). Final refinement was performed with Phenix using secondary structure restraints and noncrystallographic symmetry restraints. The structure was deposited in the PDB as
6eaq. Measurements of interfaces and buried surface area were made using the
PDBePISA utility (50). Molecular dynamics simulations of the glycosylated CD16b-Fc complex were performed using Amber16 as described (20).
Surface Plasmon Resonance (SPR) binding experiments
Binding experiments were performed as described (5). For interactions that reached a clear equilibrium, intensity values of the association phase were fitted with a binding isotherm to determine a dissociation constant for the interaction. For slower interaction where a clear equilibrium was not achieved, dissociation constants were calculated using the observed on and off rates as described (5). Values for Tables 2.1 and 2.2 include errors resulting from the least-squares fitting procedure.
Acknowledgment
We thank Dr. Daniel J. Falconer for help with X-ray crystallography and molecular dynamics simulations and Dr. Aaron Marcella for help with X-ray crystallography. Material is based upon work supported by the National Institutes of
Health under Award No. R01 GM115489 (NIGMS), the Roy J. Carver Department of
Biochemistry, Biophysics & Molecular Biology at Iowa State University, and by the 52
Department of Energy for synchrotron time at the Argonne National Laboratory
Advanced Photon Source (GUP-48455). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Department of Energy or the National Institutes of
Health.
Conflict of Interest
The authors declare that they have no conflicts of interest with the contents of this article.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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40. Radaev, S., Motyka, S., Fridman, W.-H., Sautes-Fridman, C., and Sun, P. D. (2001) The Structure of a Human Type III Fcγ Receptor in Complex with Fc. J. Biol. Chem. 276, 16469-16477 41. Mizushima, T., Yagi, H., Takemoto, E., Shibata-Koyama, M., Isoda, Y., Iida, S., Masuda, K., Satoh, M., and Kato, K. (2011) Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans. Genes Cells 16, 1071-1080 42. C, F., S, G., C, J., P, S., P, B., I, W., M, H., A, R., A, R., M, S., P, U., and J, B. (2011) Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose., Unique carbohydrate–carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose. Proc Natl Acad Sci U S A 108, 108, 12669,- 12669-12674 43. Shibata-Koyama, M., Iida, S., Okazaki, A., Mori, K., Kitajima-Miyama, K., Saitou, S., Kakita, S., Kanda, Y., Shitara, K., Kato, K., and Satoh, M. (2009) The N-linked oligosaccharide at FcγRIIIa Asn-45: an inhibitory element for high FcγRIIIa binding affinity to IgG glycoforms lacking core fucosylation. Glycobiology 19, 126-134 44. Ferrara, C., Stuart, F., Sondermann, P., Brünker, P., and Umaña, P. (2006) The Carbohydrate at FcγRIIIa Asn-162 AN ELEMENT REQUIRED FOR HIGH AFFINITY BINDING TO NON-FUCOSYLATED IgG GLYCOFORMS. J. Biol. Chem. 281, 5032- 5036 45. Ferrara, C., Grau, S., Jager, C., Sondermann, P., Brunker, P., Waldhauer, I., Hennig, M., Ruf, A., Rufer, A. C., Stihle, M., Umana, P., and Benz, J. (2011) Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose. Proc Natl Acad Sci U S A 108, 12669-12674 46. Robinson, J. I., Baxter, E. W., Owen, R. L., Thomsen, M., Tomlinson, D. C., Waterhouse, M. P., Win, S. J., Nettleship, J. E., Tiede, C., Foster, R. J., Owens, R. J., Fishwick, C. W. G., Harris, S. A., Goldman, A., McPherson, M. J., and Morgan, A. W. (2017) Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action. PNAS, 201707856 47. Summers, C., Rankin, S. M., Condliffe, A. M., Singh, N., Peters, A. M., and Chilvers, E. R. (2010) Neutrophil kinetics in health and disease. Trends Immunol. 31, 318-324 48. Subedi, Ganesh P., Hanson, Quinlin M., and Barb, Adam W. (2014) Restricted Motion of the Conserved Immunoglobulin G1 N-Glycan Is Essential for Efficient FcγRIIIa Binding. Structure 22, 1478-1488 49. Barb, A. W., Meng, L., Gao, Z., Johnson, R. W., Moremen, K. W., and Prestegard, J. H. (2012) NMR Characterization of Immunoglobulin G Fc Glycan Motion on Enzymatic Sialylation. Biochemistry 51, 4618-4626 50. Krissinel, E., and Henrick, K. (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797 57
CHAPTER 3. SITE-SPECIFIC N-GLYCAN ANALYSIS OF ANTIBODY-BINDING FC RECEPTORS FROM PRIMARY HUMAN MONOCYTES
A manuscript submitted to Molecular and Cellular Proteomics
Abstract
FcRIIIa (CD16a) and FcRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind
FcRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of
CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N- glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N- glycans at N162, however some individuals expressed CD16a V158 with ~20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites and >75% hybrid and oligomannose forms. N38 and N74 of
CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of
CD16a N-glycans isolated from monocytes included a higher proportion of 58 oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145).
Introduction
Circulating leukocytes express Fc receptors (FcRs) that trigger a protective response after encountering an antibody-coated target cell. Therapeutic monoclonal antibodies (mAbs) represent a rapidly growing class of drugs that are highly specific and generally well tolerated. The global market for mAbs, estimated at $90B in 2015, is expected to meet or exceed $140B by 2024 with many new drugs in the development pipeline (1). MAbs can treat diseases through neutralization, directly binding inflammatory molecules or eliciting the complement cascade though for the greatest efficacy the majority require FcR-mediated cell effector functions, including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis
(ADCP).
FcRIII (CD16) is the primary receptor that elicits cell-mediated immune responses and is a target for many mAbs (2-6). NK cells, macrophages and a subset of monocytes express CD16a as an integral membrane protein while neutrophils express the related glycosylphosphatidylinositol-anchored receptor CD16b (7). NK cells express only activating Fc Rs and can be activated through FcRs without priming, thus eliciting
NK cell-mediated effector function represents an important therapeutic goal (8). Other 59
CD16+ cell types including monocytes, however, respond to antibody-coated targets and contribute to clearance (9). Monocytes appeared as the dominant effector cell for B cell removal and platelet phagocytosis with little contribution from NK or T cells (10, 11).
Furthermore, increasing the strength of IgG binding by monocyte CD16a increases
ADCC, indicating that increasing the strength of the monocyte CD16a–IgG interaction may lead to greater clinical efficacy (12). These results indicate that enhancing mAb affinity for CD16a expressed on monocytes/macrophage may lead to enhanced clinical efficacy.
Monocytes are classified as classical, nonclassical and intermediate (13, 14).
The classical CD14++,CD16- phenotype represents >80% of peripheral monocytes with nonclassical (CD14+, CD16++) and intermediate (CD14++, CD16+) monocytes accounting for the remainder. Monocytes also express the FcRs CD32a and CD64.
Monocytes can differentiate into macrophages, dendritic cells and osteoclasts and adopt critical roles in tissue specific immune responses as well as wound healing (9,
15). A recent study demonstrated that monocyte ADCC activity is exclusive to CD16+ nonclassical/intermediate monocytes (16). This manuscript showed that CD16 is indispensable for proper ADCC activity while CD32a contributes as well but to a lesser extent.
The affinity between IgG and CD16a is dependent on polymorphisms and post- translational modification. The CD16a V158 allele encodes a receptor with 4-5 fold greater affinity for IgG1 than the CD16a F158 allele (17). Interestingly, clinical efficacy of glycoengineered mAbs designed to bind CD16a with higher affinity is greater in V158 homozygous individuals than F158 homozygotes, suggesting the affinity between IgG 60 and CD16a is directly correlated to clinical response (18, 19). Furthermore composition of the asparagine(N)-linked glycans also impacts the affinity of the CD16a-IgG interaction, though most studies focused on IgG N-glycan composition (2-6, 20-23).
Recent in vitro studies indicated that CD16a N-glycan composition likewise affects binding to IgG (24-26). CD16a with an oligomannose-type N-glycan bound with greater affinity to IgG1 Fc than CD16a with complex-type N-glycans (21, 23, 27). Of the five
CD16a N-glycans, the effect of N-glycan composition is primarily due to the modification at N162. Prior studies from our group identified the presence of minimally-processed endogenous CD16a glycoforms from primary NK cells and substantial donor-to-donor variability in processing at select sites (27, 52). Thus, post-translational modification plays a critical role in antibody-mediated functions, though little is known about how monocytes process CD16a.
A description of FcR processing from monocytes is expected to define what receptor forms and affinities are present at the cell surface. Isolating receptors from the surface of primary human cells represents an enormous challenge due to the small amounts of receptor in the body. We recently described the analysis of CD16a N- glycans from NK cells(52), however, monocytes represent a greater challenge. Despite the greater abundance of monocytes in the body compared to NK cells, monocytes express less CD16a than NK cells and in total contain about 20% of the CD16a compared to NK cells.
CD16a and CD32a are highly modified molecules with five and two conserved N- glycan sites, respectively. N-glycans represent the most prevalent post translational modification of excreted proteins and differences in composition result from differential 61 processing. In general, N-glycans are attached during translation and processed in the
ER and Golgi into one of three main N-glycosylation types: oligomannose, hybrid or complex-type (reviewed in (28)). Late-stage processing enzymes in the trans-Golgi modify N-glycans with late stage “caps” including sialic acid or introduce branches making highly-processed complex-type N-glycans. Alternatively, some N-glycans escape the majority of the processing enzymes; these N-glycan types include oligomannose-type which are minimally processed and hybrid-type which experience late-stage processing on only part of the glycan. N-glycosylation heterogeneity is likely dictated by abundance of the remodeling enzymes as well as enzyme localization in the
ER and Golgi network (29, 30). Cell-specific glycosylation differences have been described to be important in many biological processes, especially in development and the immune system (29, 31). Here we characterize the N-glycosylation profile of CD16a and CD32a from monocytes retained during plasma and platelet donation.
Experimental Procedures
Materials
All materials were purchased from Millipore-Sigma unless otherwise noted.
Experimental design
This study was judged by Iowa State University’s institutional review to be exempt because the donors were de-identified, the only available donor information is shown Table 3.1. Biological replicates are represented by four single donors and six donors combined into two separate pools (MoA and MoB). Lysates from certain donors 62 were pooled and processed as a single sample due to low monocyte yields or unavailability of basic information typically available for other donors. Technical replicates were not possible since cell isolation from a single donor or donor pool only provided enough material to perform a single CD16a characterization.
Protein expression
Anti-CD16 (humanized Fc) 3g8, Anti-CD16 mouse 3g8, or Anti-CD16 mouse 3g8
N297Q (heavy chain), which caused removal of N-glycosylation and was used for most of the immunoprecipitations in this study. Recombinant CD16a in HEK293F and
HEK293S were transfected and purified as previously described (32). CD16a-Man9 and
CD16a-Hybrid recombinant proteins were made by adding kifunensine and swainsonine to the culture, respectively (5 M).
Surface Plasmon Resonance
Experiments were performed and IgG1 Fc was glycoengineered as described previously (32).
Cell Isolation
Leukocyte reduction system (LRS) filters were obtained from either LifeServe
(Ames, Iowa) or the DeGowin Blood Center (University of Iowa, Iowa City) and processed within three hours of plasma or platelet donation. Cell isolations for NK cells were performed as described (27) and then frozen at -80°C except the RosetteSep
Human Monocyte Enrichment Cocktail (Stem Cell Technologies) was used to isolate 63 purified monocytes through negative selection using Lymphoprep (Stem Cell
Technologies).
Flow Cytometry
Non-specific antibody binding sites on 5 x 105 monocytes in 0.2 mL were blocked with 1 μl human serum. Then, anti-human CD14 (a mouse IgG2a, clone M5E2,
BioLegend) and/or anti-human CD16 (3G8, mouse IgG1) was added to a final concentration of 2 g/ml and cells incubated on ice for 30 minutes. Cells were then washed 2x1 ml of phosphate-buffered saline plus 0.05% sodium azide; cells were pelleted during each wash by centrifugation at 600 x g for 8 min. The secondary antibodies anti-mIgG1 (RMG1-1 conjugated to allophycocyanin, BioLegend) and anti- mIgG2a (RMG2a-62 conjugated to phycoerythin, BioLegend) were then added to a final concentration of 2 g/ml, mixed and cells incubated on ice for 30 minutes. Cells were then washed again as described above and fixed in 1% paraformaldehyde before flow cytometry analysis on the BD FACSanto (BD Biosciences) instrument. Gating of the monocyte population based on forward and side scattering was determined by excluding cell debris and smaller contaminating cells such as platelets and erythrocytes.
CD14 and CD16 gating was decided based on negative controls lacking the primary antibody.
Immunoprecipitation
Frozen cell pellets were thawed and immediately lysed using 40 L of lysis buffer
((100 mM tris(hydroxymethyl)aminomethane, 100 mM sodium chloride, 5 mM 64 ethylenediamenetetraacetic acid, 5 mM oxidized glutathione, 10 M potassium ferricyanide, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 10 mg/mL dodecylmaltoside, pH 8.0) per 1x106 cells by repeated pipetting. Lysed cells were centrifuged for 20 min at 4400 x g in 4 °C to clarify the supernatant, which was saved separately, frozen overnight, thawed on ice and centrifuged again at 4400 x g for 20 min at 4 °C. Supernatent was sonicated for 1 min at full power in a bath sonicator, 100 μL protein G sepharose (GE healthcare) was added to the supernatant and incubated for 2 h with end-over-end mixing. Resin was removed by centrifugation at 500 xg for 5 min, supernatant was moved to a new tube. Either 200 L Anti-CD16 (humanized Fc) 3g8,
Anti-CD16 mouse 3g8, or Anti-CD16 mouse 3g8 N317Q covalently coupled to aldehyde-functionalized agarose beads (Thermo Fisher Scientific) was added, and incubated overnight with end-over-end mixing at 4 °C. Resin was pelleted by centrifugation at 500 x g for 5 min and supernatant was removed and stored as the “flow through” fraction. Resin was then washed 2x 0.5 ml with lysis buffer plus 0.5 M KCl, 2x
0.5 ml lysis buffer, 2x 0.5 ml wash buffer (25 mM 3-(N-morpholino)propanesulfonic acid,
100 mM sodium chloride) and 2x 0.5 ml 50 mM ammonium bicarbonate, pH 8.0. Resin was then moved to a micro biospin column (BioRad) and eluted with 5x 0.2 ml of
(50:49.9:0.1 acetonitrile : water : trifluoroacetic acid) into 20 L 1 M ammonium bicarbonate. Samples were then pooled, frozen and lyophilized. Immunoprecipitation of
CD16a from NK cells was performed the same as described (27).
65
Western blots
The amount of recovered CD16a was estimated by western blot using recombinant CD16a as a standard. Western blots were performed as described (27).
CD16a glycosidase treatment
CD16a from primary NK cell and monocyte was immunoprecipitated as mentioned above except CD16a was not eluted after the wash steps and digestions were performed on CD16a bound to 3G8 agarose resin. Endo F1 was expressed using a plasmid provided by Dr. Kelley Moremen (University of Georgia) and isolated as mentioned previously (33). CD16a-loaded 3G8 agarose resin was diluted using 250 mM sodium phosphate buffer, pH 5.5. Endo F1 was added and incubated at 37 °C for 1.5 h.
The reaction was stopped by boiling for 5 min in the presence of SDS-PAGE loading buffer (BioRad). PNGase F digestion was performed by following the manufacturer’s protocol (New England Biolab). Briefly, resin was boiled in presence of denaturing buffer before adding NP-40, reaction buffer and PNGaseF. The reaction was incubated at 37
°C for 4 h and was stopped by boiling the samples in presence of SDS-PAGE gel loading buffer. Soluble CD16a (sCD16a) from NK cell and monocytes was obtained by incubating isolated NK cell and monocytes at 37°C for 1 h at a cell density of 10×106 cells/ml in RPMI 1640 (Thermo Scientific) medium supplemented with 1% fetal bovine serum (VWR), 1 g/ml phorbol 12-myristate 13-acetate and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (Millipore Sigma). The cells were removed by centrifugation and the supernatant frozen until sCD16a was immunoprecipitated using the method described above. Negative controls for each reaction were treated as digested samples 66 except enzymes were not added. Recombinant CD16a (250 ng) expressed in HEK293F or HEK293S served as a positive control. Boiled samples were directly loaded on 12%
SDS-PAGE gels and analyzed using antiCD16 western blot.
CD16a proteolysis
CD16a was resuspended in 100 l of 50 mM ammonium bicarbonate, pH 8.0 and heat denatured at 90 °C for 5 min and cooled on ice. A 1:40 (w:w) ratio of chymotrypsin and 1:30 (w:w) of Glu-C (Promega) was added and the solution incubated at 25 °C overnight. Fresh chymotrypsin and Glu-C was added and the solutions incubated for 3 h. Chymotrypsin is specific for FYW with lower specificity for ML while Glu-C is specific for E. Both enzymes cut at the C-terminal of the mentioned amino acid residue.
Following proteolysis, dithiothreitol was added to a final concentration of 5 mM and the solution incubated for 1 h at 37 °C. Iodacetamide was then added to a final concentration of 15 mM and the solution incubated in the dark at RT for 1 h. The sample was then centrifuged for 2 min at 9600 x g, the supernatant transferred to a new tube, frozen at -80 °C and lyophilized. Lyophilized samples were resuspended in 10 l of water and then glycopeptides were enriched using the ProteoExtract Glycopeptide
Enrichment Kit following the manufacturer’s guidelines to separate peptides from glycopeptides. Isolated glycopeptides were then frozen, lyophilized, resuspended in a solution containing 5% acetonitrile and 0.1% formic acid before MS analysis.
Peptides were desalted using ethyl acetate: a glass tube was washed with 200 ml ethyl acetate. Water saturated ethyl acetate was made by mixing 5 ml ethyl acetate with 625 l water in the washed glass tube. 1 ml of the upper phase was added per 100 67
l sample. Samples were vortexed for 1 min, centrifuged for 12,500 x g and the upper layer was removed and discarded. This washing procedure was repeated 3 times.
Residual ethyl acetate was removed from the top under a nitrogen stream. Isolated peptides were then frozen, lyophilized, resuspended in a solution containing 5% acetonitrile and 0.1% formic acid in water prior to MS analysis (34).
Mass Spectrometry
Sample (5 L) in buffer A (5% acetonitrile, 94.9% water and 0.1 % trifluoroacetic acid) was loaded onto a 75 mm x 20 cm column packed with C18 Zorbax resin (3 or 5
m) equipped with a pulled glass emitter using an EASY-nLC 1200 nanopump (Thermo
Fisher Scientific) in line with a Q-exactive Hybrid Quadrupole-Orbitrap Mass
Spectrometer (Thermo Fisher Scientific). The column was briefly washed with buffer A.
Analytes were eluted at flow rate of 300 nl/min with an increasing linear gradient of buffer B (99.89% acetonitrile, 0.01% formic acid, 0.1% trifluoroacetic acid) from 0 to
35% over 60 min. Buffer B was then increased to 70% for 10 min and then 100% for 10 more min before returning to 0% B. In between experimental samples, buffer A (20 l) was loaded on to the column and eluted as a “blank”. A peak observed at 600 m/z in the
MS2 spectra represents carryover from a calibration standard.
Data Analysis
Raw data files were initially analyzed with Byonic (Protein Metrics). The search parameters included Cleavage site (FYWEL), Cleavage side (C-terminal), Digestion specificity (Semi specific (slow)), Missed cleavages (2), Precursor mass tolerance (5 68 ppm), Fragment mass tolerance ( 0.05 Dalton), Recalibration (lock mass) (none), maximum precursor mass (5,000), Precursor and mass charge assignments (Compute from MS1), Maximum # of precursors per scan (10), smoothing width (0.01 m/z) and the following modifications included (in addition to N- and O-glycan databases):
Carbamidomethyl / +57.021464 @ C | fixed; Deamidated / +0.984016 @ N | common1;
Acetyl / +42.010565 @ Protein N-term | rare1; Phospho / +79.966331 @ C, D, H, K, R,
S, T,Y | common2; HexNAc / +203.079373 @ N,S,T | common1. Hits were then manually validated and additional peaks not identified by Byonic were found.
Glycopeptide annotations were made in Glycoworkbench Version 2.1 (35). Annotated
MS2s for each species are shown in a Supplemental PDF and a recent characterization of this purification and analysis strategy using NK cells (52). Species without clear MS2 spectra were assigned if they showed retention times and predictable differences from known species (ie 162.05 Da). N-glycan structures indicate composition only without linkage information elucidated. Analyte intensities were measured from MS1 spectra using scripts in R based on retention time windows defined for each species (52). This approach measured the intensity of as many as seven isotopologue peaks for each glycopeptide. Isobaric species were not differentiated.
Donor genotyping
Blood retained in the tubing of the apheresis filters (~50 L) was centrifuged at
10,000 x g for 10 min and the cell pellet and serum fractions were stored separately at -
80 °C. The RNA from the cell pellet was isolated using the Trizol Reagent as directed by manufacturer (Thermo Fisher Scientific). cDNA was prepared from the isolated RNA 69 using the High Capacity RNA to cDNA as directed by the manufacturer (Thermo Fisher
Scientific). The full FCGR3A ORF including region coding for amino acid 158 in the final protein was then amplified using AccuPrime PFX with forward (5’-
CAGTGTGGCATCATGTGGCAG-3’) and reverse (5’-TTTGTCTTGAGGGTCCTTTCT-
3’) primers. Resulting DNA was purified with a Gel Extraction Kit (Qiagen) and submitted for Sanger Sequencing at the Iowa State Sequencing Facility.
Results
Apheresis filters entrap monocytes
We isolated primary human monocytes from apheresis filters following plasma or platelet donation to define specific features of CD16a expressed by monocytes.
Apheresis filters provided an average of 4 x 108 monocytes per donor following negative selection (Table 3.1). Sequencing cDNA from each donor’s leukocytes revealed that the majority of donors expressed both the V158 and F158 alleles, as expected (Table 3.1).
Flow cytometry indicated a high percentage of cells (82.3 - 97.6%) expressed CD14 in the expected proportion of “classical” CD14+ CD16dim to “non-classical” CD14dim CD16+ monocytes (83-85% and 15-17%, respectively; (13, 14, 36)). Individual donors also displayed variation in the level of CD16 staining for the CD16+ monocytes.
CD16a N-glycan modifications
Previous studies indicated CD16a N-glycosylation composition affects binding to
IgG1, however it was unknown if forms that bind with high affinity in vitro are present on circulating monocytes (23, 27). We analyzed six monocyte CD16a samples, four 70 isolated from single donors and two as pools of three donors (Table 3.1). Data from these donors revealed 133 unique CD16a glycopeptides that contained a total of 77 unique N-glycans (Table 3.2). Our analysis revealed only N-glycan modifications. We also characterized non-modified peptides for a total of 84% coverage of the peptide backbone (Figure 3.1C).
Table 3.1. Donor information ABO Rh Date of Monocytes CD16a Donor Gender Age group factor collection isolated x106 genotype
MoA (pool)
Mo01 n.d. n.d. n.d. n.d. 6/9/2017 200 V/F
Mo03 n.d. n.d. n.d. n.d. 6/9/2017 329 V/F
Mo05 n.d. n.d. n.d. n.d. 6/9/2017 351 V/V
MoB (pool)
Mo06-i n.d. n.d. n.d. n.d. 6/9/2017 580 V/F
Mo06-ii F 73 AB + 3/16/2017 280 F/F
Mo06-iii F 35 AB + 4/6/2017 139 V/F
Mo02 F 17 B + 6/9/2017 200 V/V
Mo04 n.d. n.d. n.d. n.d. 6/9/2017 362 F/F
Mo11 M 61 A + 6/8/2018 406 V/F
Mo13 F 40 O + 8/28/2018 303 F/F
Mo15 M 27 O + 8/28/2018 588 V/F n.d. = not determined
71
Figure 3.1. CD16a N-glycosylation composition varies by site and donor on monocytes. A. CD16a contains 5 N-glycosylation sites on the extracellular domain. These sites are modeled from the most abundant forms found on monocytes; the ribbon diagram and surface rendering are based on known structures of CD16a determined by X-ray crystallography. Carbohydrate residues are modeled as cartoons using the SNFG convention and roughly scaled to size (45). B. The relative abundance of each N-glycan type for the four individual donors and two donor pools as specified in Table 3.1. n.a. – not applicable. C. ESI-MS/MS identified peptides and glycopeptides covering 84% of the CD16a sequence. Glycosylated Asn residues are shown in red. The V/F158 amino acid residue is labeled in blue.
Subtle donor-dependent variation in N162-glycosylation
The N162 glycopeptides also contain F158 or V158, linking the CD16a allele with processing at N162. The glycopeptides identified for each donor matched those expected based on cDNA sequencing (Table 3.2). The N162 glycopeptides produced ions with relatively high intensity and high quality MS2s for most species as shown in
Figure 3.2. HCD fragmented ions confirm composition of the ion, in particular through monosaccharide species specific for sialic acid (292.10 Da or 274.09 Da (-H2O)) and 72
HexNAc (204.09 Da), disaccharide species including HexNAc-Hexose (366.14 Da), and trisaccharide species including HexNAc-Hexose-Sialic acid (657.24 Da). A peak corresponding to the peptide + HexNAc is a common diagnostic glycopeptide and also one of the most intense as noted previously (43). Most of these ions contain a deoxyhexose residue and fragments corresponding to the peptide + HexNAc + deoxyhexose and are likely core fucosylated, though we cannot eliminate the possibility of branch fucosylation because isomeric species were likely not separated during chromatography.
Table 3.2. Glycopeptides identified in this study.
Mo02 MoA MoB Mo11 Mo13 Mo15 total unique site # Peptide (V/V) (V/F) (V/F) (V/F) (F/F) (V/F) glycopeptides
1 KCQGAYSPEDNSTQW 6 2 5 8 n.d. 2 11
N38 2 SPEDNSTQW 3 3 n.d. 7 n.d. n.d. 9
1 FHNE 17 16 10 14 11 15 21
N45 2 FHNESLISSQASSY 5 18 18 8 3 10 28
N74 1 RCQTNLSTLSDPVQLE 16 7 5 3 4 3 24
N162 1 CRGLVGSKNVSSE 10 20 8 15 n.a. 6 21 (V158)
N162 2 GSKNVSSE n.a. 1 6 2 2 4 6 (F158)
N169 1 TVNITITQGL 5 7 3 2 2 2 9
2 TVNITITQGLA n.d. 1 2 3 2 n.d. 4
total unique glycans: 77 total glycopeptides: 133
n.a = not applicable
n.d = not detected 73
The predominant N162 glycans, averaged across all donors and allotypes, are summarized in Figure 3.2 with a core-fucosylated monosialylated complex-type N- glycan representing the most abundant species, though other forms also appear including complex-type biantennary, triantennary and hybrid-types. Other low abundant species include tetrantennery complex-type as well oligomannose-type N-glycans.
Interestingly, four datasets revealed small amounts of oligomannose-type N162-glycans
(<10%). Overall, we determined that the N162-(F158)-glycan from these six datasets was 99.7% complex-type and 0.3% hybrid-type while the N162-(V158)-glycan was
88.9% complex-type, 7.1% hybrid-type and 3.9% oligomannose-type N-glycans.
CD16a V158 with hybrid or oligomannose-type N162-glycans exhibits greater affinity for IgG1 Fc
The appearance of under-processed N162 glycoforms from some donors is noteworthy because CD16a V158 with oligomannose-type N162 glycans binds IgG1 Fc with greater affinity in vitro, suggesting the monocytes from some donors express
CD16a with enhanced affinity (20, 21, 23, 27). However, it is unclear how hybrid-type N- glycans impact affinity or if the CD16a F158 allotype displays a similar enhancement.
SPR binding studies between IgG1 Fc and CD16a expressing complex-type (CT), Man9 or hybrid-type N-glycans indicate an affinity enhancement when CD16a has Man9 or hybrid-type as compared to complex-type (Table 3.3). These results are comparable to previous measurements from our lab, though the absolute difference in enhancement due to the presence of a oligomannose-type N-glycans is reduced (20, 21, 23, 27). 74
Likewise, these are consistent with previous data showing the negative impact of Fc fucosylation on affinity for all three V158 glycoforms. In this experiment, the hybrid-type
N-glycans increased CD16a V158 binding to IgG1 Fc by 1.4-2.3 fold, while oligomannose N-glycans increased affinity by 2.5-2.9 fold as compared to CD16a V158-
CT. As expected, CD16a F158 bound IgG1 Fc with lower affinity than the V158 allotype
(17). Surprisingly, receptor glycan composition did not appear to impact binding affinities of the CD16a F158 allotype.
Figure 3.2. The CD16a N162-glycan is predominantly a monosialylated biantennary complex-type with some hybrid/oligomannose. The relative abundance represents the averages of each species from datasets originating from individual donors and donor pools and the pie charts represent the average abundance of each N-glycan type. A. Relative abundance of F158 glycopeptides (1 and 7). B. Relative abundance of glycopeptides for a single V158-specific peptide. C. A representative MS2 spectrum for most abundant N162 F158 glycopeptide. D. A representative MS2 spectrum for most abundant N162 V158 glycopeptide. Cartoons indicate one possible glycan configuration consistent with the composition, isobaric ions are not distinguished.
75
Table 3.3. Equilibrium dissociation constants (KD) from SPR analysis of the CD16a and IgG1 Fc interaction (Errors represent errors of fit to the Langmuir binding isotherm; this experiment is representative of three independent experiments). IgG1 Fc G0F IgG1 Fc G0
Receptor KD (nM) error KD (nM) error
CD16a V158-CT 500 30 100 10
CD16a V158-Man9 170 20 40 10
CD16a V158-Hybrid 220 20 70 10
CD16a F158-CT 1500 100 240 40
CD16a F158-Man9 1300 200 200 70
CD16a F158-Hybrid 1000 100 200 10
Restricted processing and variability at N45
N45 glycosylation shows the most restricted processing and greatest variability compared to the other four CD16a N-glycosylation sites. Two N45 peptides contained the N45-glycan (Table 3.2), N45 peptide 1 is the expected degradation product and peptide 2 is the product of a missed Glu-C cleavage. A hybrid-type N-glycan with a sialic acid residue, three mannose residues that lacks fucosylation is the most predominant species found in all six datasets (Figure 3.3). N45 glycan exhibited substantial variability between donors with a range of 1.0 - 50.2% complex types, 36.0 -
64.0% hybrid types and 1.7 - 58.8% for oligomannose types. MS2 fragmentation of hybrid-type N-glycan with one fucose and one sialic acid as well as an oligomannose- type glycan are illustrated in Figure 3.3. Diagnostic fragments in the MS2 spectra include hexose (163.06 Da), HexNAc (204.09 Da) and HexNAc + hexose (366.14 Da) appeared in many MS2 spectra along with peptide fragmentation including y- and b- series ions. 76
Figure 3.3. N-glycosylation at N45 shows restricted processing with mostly hybrid and oligomannose N- glycans. A. Schematic showing the location of N45. B. Charts showing the average abundance of each N-glycan type. C. The most abundant N45-glycan species identified, ranked according to average abundance. The intensities of N45 glycopeptides 1 and 2 were combined for this analysis. MS2 fragmentation for the most abundant hybrid (D.) and oligomannose-type N-glycans(E.).
Extensive modification with LacNAc units at N38 and N74
N-glycans at the N38 and N74 sites displayed exclusively highly processed complex-type N-glycans (Figure 3.4). Repeats of a hexose-HexNAc moiety define the predominant features of these species, though sialic acids and fucosylation are also present. The most intense ions differed by the number of hexose-HexNAc units and contained 2-5 sialic acids. Analysis of the monoisotopic peaks from the parent ions in the MS1 dimension provided differentiation of species to determine the presence of two 77 fucose residues versus one sialic, thus there are at most 4 sialic acids at these two sites. Due to the large size of these ions and overall lower intensity compared to the
N45 and N162 glycopeptides, MS2 fragmentation proved much weaker for these species and only showed the diagnostic peptide + GlcNAc ion for N38 and N74 (data not shown).
Figure 3.4. Processing of the N38, N74 and N169 N-glycans. N-glycans at N38 (A.) and N74 (B.) are exclusively highly processed forms with LacNAc repeats. C. N-glycosylation at N169 is predominantly a complex-type biantennary form with some tetra-antennary complex-types as determined by combining intensities of peptide 1 and 2.
N169-glycans are predominantly complex-type
Complex-type N-glycans exclusively occupied N169 in all donors (Figure 3.1). A complex-type biantennary with one fucose and one sialic acid was the most 78 predominant N169-glycan, the same predominant form found at N162 (Figure 3.2). The
ADAM17 cleaved peptide (N169 peptide 2) was identified but at a much lower intensity when compared to the expected Glu-C and chymotrypsin peptide (N169 peptide 1)
(Table 3.2), though this may result from non-specific proteolysis during peptide purification.
Monocytes process CD32a differently than CD16a
Monocytes also express CD32a that contains two N-glycosylation sites and glycan profiles that are unlike CD16a. Two pooled datasets (MoA and MoB) included
CD32a peptides because we purified CD16a from these samples using 3G8 (a mouse
IgG1) that contained an Fc N297-glycan that captured CD32a, unlike the single-donor datasets purified over immobilized 3G8 without Fc N-glycosylation that didn’t bind
CD32a (44). Analysis of the peptides from these datasets provided 61% sequence coverage of CD32a (Figure 3.5). Of these two datasets, MoB produced N64 and N145
N-glycopeptide ions with high intensity in the MS1 dimension and high quality MS2 spectra (Figure 3.5). N-glycans at these sites were predominantly complex-type biantennary species with a core fucose residue and two sialic acids. MS2 fragmentation confirmed these assignments (Figure 3.5E and F). MoA provided only a single N64 glycopeptide that matched the predominant N64 species from MoB. Similarly, the most abundant N145 species from both datasets proved identical. The MoA dataset included
7% oligomannose forms and 2% hybrid forms unlike the MoB dataset that revealed no oligomannose forms and 17% hybrid forms (data not shown). These data indicate the 79 possibility for a surprising degree of variability at the N145 site and stark differences in processing compared to CD16a from the same donor pools.
Figure 3.5. Purified CD32a contains predominantly complex-type biantennary N-glycans. A. CD32a contains 2 N-glycosylation sites on the extracellular domain. These sites are modeled from the most abundant forms found on monocytes; the ribbon diagram and surface rendering are based on known structures of CD32a determined by X-ray crystallography. The most abundant N-glycans at N64 (B) and N145 (C). D. ESI-MS/MS identified peptides and glycopeptides covering 61% of the CD32a sequence. Glycosylated Asn residues are shown in red. Representative MS2 spectra for the most abundant N- glycoforms at N64 (D.) and N145 (E.) Data in B, C, E & F are from the MoB pool. 80
Discussion
We defined the composition of CD16a N-glycans isolated using primary human monocytes recovered from apheresis filters. These data indicated that location impacted
CD16a N-glycan composition to a greater degree than other variables including donor age, genotype, etc. (Figure 3.1B, Table 3.2). In general, the CD16a N38 and N74- glycans are exclusively of a highly-processed complex-type with N-acetyllactosamine repeats, N169 are complex-type, N162 are predominantly complex-type with some less- processed forms present in some donors, and N45 contains a mixture of complex, hybrid and oligomannose types. This analysis reports increased sensitivity compared to the previous analysis of CD16a from the NK cells of single donors reported by our group
(52). Though monocytes are more abundant in the peripheral blood of most donors, monocytes express less CD16a than a typical NK cell.
The initial comparison of CD16a from monocytes and NK cells revealed differences that are attributable to N-glycan composition (data not shown). Surprisingly,
EndoF1 digested NK cell CD16a but not monocyte CD16a. This is likely due to differences in N45-glycan composition; monocyte N45 contains a much higher proportion of smaller hybrid types that lack a fourth mannose residue (likely the 1-
3Man on the 1-6Man branch) that is required for EndoF1 activity (45) and is found on the NK cell CD16a N45 N-glycans (52).
Restricted processing at N45 is not unique to monocyte CD16a (Figure 3.2). NK cell CD16a N-glycosylation displays more restricted processing than recombinant
CD16a expressed in HEK293F cells (27). A recent study found CD16a N45 on NK cells 81 displays predominantly hybrid-type (52) and hardly any oligomannose forms unlike
CD16a from monocytes reported here which has mostly hybrid and oligomannose-type.
Furthermore, CD16b, the highly related FcR expressed by neutrophils contains predominantly oligomannose and minimal hybrid-type N45-glycans unlike CD16a from both NK cells and monocytes (43, 46). CD16 N45 N-glycosylation processing can be summarized as NK cell > monocyte > neutrophil. NMR and molecular dynamics simulations indicate the CD16 N45 N-glycan makes intramolecular contacts with the polypeptide leading to decreased mobility of the N-glycan (21, 22). Thus, restricted processing of N45 likely results from carbohydrate-polypeptide contacts that reduce accessibility to glycosyl transferases and glycosidases.
CD16 N162 processing on NK cells varied by donor though neutrophils or monocytes expressed receptor with much less variability at this site (Figure 3.2; (43,
46,52)). Processing at the remaining three sites is more directly comparable between the three cells types. Gene microarray data indicate that monocytes express the biosynthetic machinery for multiantennary branching of N-glycans including N- acetyllactosamine structures (47). MGAT5 catalyzes addition of the fourth branch
GlcNAc residue to the 6-OH of the 1-6Man residue of a triantennary complex-type N- glycan, providing the likely site for LacNAc polymerization (48). Interestingly, Galectin 3 binds poly-LacNAc motifs on N-glycans and regulates T cell receptor clustering and cell activation (49, 50). It remains unclear if CD16a LacNAc motifs on NK cells, monocytes or neutrophils adopt similar roles.
This cell isolation and purification strategy proved sufficient to determine the unique distribution of N-glycoforms at each of the five CD16a N-glycan sites and 82 provided a comparable analysis of CD32a from the same cells. Interestingly, the two N- glycosylation sites on CD32a, N64 and N145, were predominantly complex-type biantennary with two sialic acids and one fucose, unlike any of the major forms on the five CD16a sites. Thus, these monocytes processed CD16a and CD32a N-glycosylation differently. Because the purification targeted CD16a, it is possible that stronger binding
CD32a glycoforms preferentially bound the mIgG1 and are thus enriched in these datasets, however this appears unlikely because binding differences of CD32a glycoforms for human IgG1 Fc were not identified (23). Saggu and coworkers recently characterized CD32a N-glycans purified from neutrophils and identified the identical predominant N64-glycan but a high percentage of under-processed N145 glycans including oligomannose and hybrid-types (44). These authors found sialylated CD32a interacted with Mac1 which downregulated CD32a activity implicating CD32a N- glycosylation as functionally relevant mediators of neutrophil activation. Interestingly, monocytes reportedly downregulate Mac-1 expression after incubation with IgG, CD32a
N-glycans may perform a comparable role on monocytes (51).
In summary, these data provide insight into how an individual donor modifies individual N-glycosylation sites on two different FcRs recovered from a single cell type.
Differential modifications potentially provide an individual with the ability to modify the activation threshold by controlling receptor affinity, and this approach affords the ability to characterize FcRs from circulating monocytes using cells isolated from a single donor.
83
Acknowledgment
We thank donors and staff at the DeGowin Blood Center (Iowa City, IA) and
LifeServe Blood Center (Ames and Des Moines, IA) for providing materials. We also thank Joel Nott for the MS ananlysis (ISU Protein Facility) and Dr. Sean Rigby (ISU
Flow Cytometry Facility) for performing the flow cytometry measurements. Kashyap
Patel for help with cell isolation and flow cytometry. This material is based upon work supported by the National Institutes of Health under Award No. R01 GM115489
(NIGMS) and R21 AI142122 (NIAID). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institutes of Health.
Data availability
Raw data available at: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp
Username: Adam_Barb_Lab_1
Password: password1
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(FcgammaRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One 10, e0121788 41. Edberg, J. C., Barinsky, M., Redecha, P. B., Salmon, J. E., and Kimberly, R. P. (1990) Fc gamma RIII expressed on cultured monocytes is a N-glycosylated transmembrane protein distinct from Fc gamma RIII expressed on natural killer cells. J Immunol 144, 4729-4734 42. Edberg, J. C., and Kimberly, R. P. (1997) Cell type-specific glycoforms of Fc gamma RIIIa (CD16): differential ligand binding. J Immunol 159, 3849-3857 43. Washburn, N., Meccariello, R., Duffner, J., Getchell, K., Holte, K., Prod'homme, T., Srinivasan, K., Prenovitz, R., Lansing, J., Capila, I., Kaundinya, G., Manning, A. M., and Bosques, C. J. (2019) Characterization of Endogenous Human FcgammaRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation. Mol Cell Proteomics 18, 534-545 44. Saggu, G., Okubo, K., Chen, Y., Vattepu, R., Tsuboi, N., Rosetti, F., Cullere, X., Washburn, N., Tahir, S., Rosado, A. M., Holland, S. M., Anthony, R. M., Sen, M., Zhu, C., and Mayadas, T. N. (2018) Cis interaction between sialylated FcgammaRIIA and the alphaI-domain of Mac-1 limits antibody-mediated neutrophil recruitment. Nat Commun 9, 5058 45. Varki, A. (2017) Biological roles of glycans. Glycobiology 27, 3-49 46. Yagi, H., Takakura, D., Roumenina, L. T., Fridman, W. H., Sautes-Fridman, C., Kawasaki, N., and Kato, K. (2018) Site-specific N-glycosylation analysis of soluble Fcgamma receptor IIIb in human serum. Sci Rep 8, 2719 47. Trottein, F., Schaffer, L., Ivanov, S., Paget, C., Vendeville, C., Cazet, A., Groux- Degroote, S., Lee, S., Krzewinski-Recchi, M. A., Faveeuw, C., Head, S. R., Gosset, P., and Delannoy, P. (2009) Glycosyltransferase and sulfotransferase gene expression profiles in human monocytes, dendritic cells and macrophages. Glycoconj J 26, 1259- 1274 48. Sasai, K., Ikeda, Y., Fujii, T., Tsuda, T., and Taniguchi, N. (2002) UDP-GlcNAc concentration is an important factor in the biosynthesis of beta1,6-branched oligosaccharides: regulation based on the kinetic properties of N- acetylglucosaminyltransferase V. Glycobiology 12, 119-127 49. Nabi, I. R., Shankar, J., and Dennis, J. W. (2015) The galectin lattice at a glance. J Cell Sci 128, 2213-2219 50. Demetriou, M., Granovsky, M., Quaggin, S., and Dennis, J. W. (2001) Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature 409, 733-739 51. Fernandez-Calotti, P. X., Salamone, G., Gamberale, R., Trevani, A., Vermeulen, M., Geffner, J., and Giordano, M. (2003) Downregulation of mac-1 expression in monocytes by surface-bound IgG. Scand J Immunol 57, 35-44 88
52. Patel, K. R., Nott, J. D. & Barb, A. W. Primary human natural killer cells retain proinflammatory IgG1 at the cell surface and express CD16a glycoforms with donor- dependent variability. Mol. Cell Proteomics (2019). doi:10.1074/mcp.RA119.001607 89
CHAPTER 4. RESTRICTED PROCESSING OF CD16A / FC RECEPTOR IIIA N- GLYCANS FROM PRIMARY HUMAN NK CELLS IMPACTS STRUCTURE AND FUNCTION
A manuscript published in the Journal of Biological Chemistry.
Patel, K. R.*, Roberts, J. T.*, Subedi, G. P.* & Barb, A. W. Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function. J. Biol. Chem. jbc.RA117.001207 (2018). doi:10.1074/jbc.RA117.001207 An * after author name indicates equal author contribution
Initials at the end of subheading titles indicate contributions to that section
Abstract
CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer(NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue.
Therapeutic monoclonal antibodies(mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome, however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates(N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid(22%) and oligomannose N-glycans(23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans(82%).
Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via co-expression with the Fc ε receptor –chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell-derived CD16a but 90 yielded N-glycoforms different from NK cell-derived CD16a and recombinant soluble
CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans.
The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein’s structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines
CD16a N-glycan composition and antibody binding affinity.
Introduction
Antibodies activate a diverse set of protective responses following the recognition of a previously-encountered epitope. Therapeutic monoclonal antibodies (mAbs) circumvent prior exposure to induce a protective response against diseased tissue by eliciting the same protective mechanisms, including antibody-dependent cell mediated cytotoxicity (ADCC) through binding Fc receptors (FcRs), and represent a major drug class worth tens of billions of dollars each year in global sales. MAbs are predominantly built from immunoglobulin G (IgG) and a large proportion bind FcRs expressed on a range of leukocytes to initiate target destruction. However, very little is known about the composition of FcRs from native leukocytes and how this composition affects IgG binding and thus treatment efficacy. 91
ADCC is predominantly performed by natural killer (NK) cells (1), cytolytic lymphocytes present as 1-6% of circulating leukocytes (2,3) that express
FcRIIIa/CD16a as the predominant FcR. The activating role of CD16a in ADCC by NK cells is well established (1). Indeed, investigations of rituximab, trastuzumab and cetuximab therapeutics demonstrated the importance of CD16a on NK cells for success
(4).
Improved therapeutic mAb efficacy can be achieved by enhancing the mAb-
CD16a interaction. In-vitro binding studies demonstrated the less common CD16a V158 allotype binds IgG1 with ~5 fold greater affinity compared to CD16a F158 (5).
Furthermore, increased binding leads to an increased NK cell response and increased
ADCC (6-9). Current advances in mAb therapy are targeting optimization of NK cell
ADCC activity by Fc glycoengineering to improve CD16a binding (4). These efforts include targeted modifications to the asparagine-linked carbohydrate (N-glycan) attached to N297 of the IgG1 heavy chain (9,11). A glycoengineered mAb performed better in the clinic when compared to a non-optimized version (4,12).
N-glycans contribute to protein function and are targets for new therapies or enhancing existing therapies. N-glycan processing occurs during protein secretion. The types and extent of modifications are not template driven, thus, it is not currently possible to predict N-glycan composition from DNA sequence information. Following the en bloc transfer of a 14-residue precursor N-glycan as directed by the presence of a N-
X-S/T sequon where X is any residue except proline, the glycoprotein associates with protein-folding specific chaperones, and if properly folded, continues through the ER and Golgi-mediated secretory pathway to encounter remodeling glycosylhydrolase and 92 glycosyltransferase enzymes (Figure 4.1). Though many factors contributing to N- glycan composition on any given protein remain undefined, it is generally believed that modifying enzyme expression, protein structure and the presence or absence of a membrane anchor affect N-glycan composition in the secreted protein (13). N-glycans that experience minimal processing emerge from the cell as oligomannose forms
(Figure 4.1B). More highly processed N-glycans may also include hybrid forms and the predominant complex-types found on cell surfaces and circulatory serum proteins.
Additional to folding, N-glycans promote secretion, protection against proteolysis, reduce antigenicity, extended serum half-life, and contribute specific epitopes for receptor binding as well as impact protein structure and function (14-16).
Figure 4.1 (AWB). Membrane-anchored CD16a binds IgG antibodies. A. CD16a (orange ribbon) contains five potential N-glycosylation sites in the extracellular domains (orange spheres). B. N-glycan processing occurs in the ER and Golgi following transfer of a large glycan en bloc to the protein and produces oligomannose, hybrid and complex-type N-glycans. Individual carbohydrate residues are colored according to the SNFG convention (14). 93
The CD16a sequence encodes five N-glycosylation sites, though little is known about either the N-glycan composition or how CD16a N-glycans impact function. Edberg and Kimberly reported that NK cell CD16a contained a high degree of oligomannose- type N-glycans using immobilized lectin chromatography and found little evidence for complex or hybrid types (17). This characterization was in stark contrast to later mass spectrometry-based reports on the glycosylation of recombinant CD16A which identified predominantly complex-types with low percentages (2-5%) of oligomannose N-glycans
(18-21), however, these recombinant expressions did not include the membrane anchor and intracellular domains, as well as the coexpressed Fc receptor - or CD3 - chains that may influence N-glycan processing. It is well known that N-glycan composition can influence protein function (15,22-24), with some evidence indicating composition might affect the antibody-binding function of CD16a (21). Thus, the in vitro analyses of monoclonal antibody binding by CD16a should include proper N-glycoforms for representative results. At this point, there has not been a report on the high-resolution
N-glycan composition of CD16a from primary human leukocytes.
Access to a sufficient quantity of primary human tissue represents a significant barrier to high-resolution composition studies because CD16a is expressed by a limited subset of leukocytes, including natural killer cells and CD16a+ monocytes that constitute
1-6% and 0.4-2.4% of circulating leukocytes, respectively (3,25). We predict that it would require as much as 1.25 L of human blood to recover a minimum of 100 x106 natural killer cells and 1 g of CD16a from a single donor to provide sufficient material for characterization. It is untenable to harvest one-quarter or more of a donor’s blood 94 volume for obvious reasons. The adoption of apheresis systems by blood donation centers to collect plasma, platelets and other blood components provides a valuable source of viable primary human lymphocytes from peripheral blood through leukocyte reduction system (LRS) filters that are normally discarded following donation (26). In this manuscript we describe a method to purify CD16a from primary human NK cells from a single donor to analyze N-glycan composition.
Results
NK cell isolation and CD16 purification
Light scattering of isolated NK cells combined with CD56 and CD16 staining assessed by flow cytometry revealed >90% cell purity, >87% viability and a high degree of CD16 expression (Figure 4.2). A blot of NK cell lysate probed with an anti-CD16 antibody showed a band from 53-59 kDa (Figure 4.2C). Despite differences in CD16 staining intensity measured from more than a dozen donors, NK cell lysate repeatedly showed indistinguishable CD16 migration characteristics (data not shown). The mobility of the NK cell CD16 band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiment increased following digestion with PNGaseF, an enzyme that specifically removes N-glycans. However, the resulting two bands at
~33 kDa are still larger than expected for the 27 kDa CD16a polypeptide (Figure 4.2C).
This treatment indicates that N-glycans retard CD16 mobility in a polyacrylamide gel.
The CD16 identified here is consistent with CD16 from multiple previous reports based on migration in SDS-PAGE (27-30). 95
Figure 4.2 (JTR/KRP). Representative NK cell isolation and CD16a purification. A. Light scattering and B. double-antibody staining of negatively-selected NK cells. Isotype and negative staining controls are shown in Figure S1. C. Anti-CD16 western blot of PNGase F-digested CD16a shows an increase in mobility following N-glycan removal. D. Anti-CD16 western blot of CD16a purification from an NK cell lysate compared with recombinant CD16a truncated at the transmembrane domain (HEK293F).
A monoclonal anti-CD16 mouse IgG1 antibody, 3G8 (31), is widely used for flow cytometry but not for western blot applications because CD16 denaturation destroys the epitope (data not shown). Thus, 3G8 is a good candidate to precipitate folded and processed CD16 from cell lysates at a preparative scale. Our purification scheme first lyses NK cells in detergent, followed by incubation with protein G resin to remove IgG that may obscure the CD16 epitope recognized by the 3G8 antibody, adsorption to a 96
3G8-agarose resin, extensive washing to remove material that weakly interacts with the resin, and finally elution with 45:55:0.1 water:ACN:TFA. This procedure showed clear depletion of CD16 from the NK cell lysate and enrichment in the elution fraction with recovery of ~1 g CD16 / donor (Figure 4.2D).
CD16a validation with ESI-MS/MS
The observed mobility of CD16 from primary human NK cells in SDS-PAGE gels is similar to previous reports and likely represents similar material, though peptide sequencing or other comparable techniques were not previously applied for validation.
Thus, we applied high-resolution peptide sequencing to verify the immunoprecipitated material. Purified and trypsin-digested CD16a from a single donor’s NK cells generated multiple polypeptide fragments in an HPLC(C18)-ESI-MS/MS experiment that fit with high confidence to 47% of the CD16a sequence (Figure 4.3). One peptide revealed this donor expressed the predominant CD16a F158 allele (Figure 4.3D). The donor allotype was also confirmed by cDNA sequencing (data not shown). Peptides containing predicted CD16a N-glycosylation sites, transmembrane or intracellular regions were not detected but the observed peptides were enough to confirm CD16a identity and found no other peptides. It is possible that the immunoprecipitated material includes low levels of contaminating proteins. To address this potential shortcoming we analyzed PNGase-
F treated peptides from a single donor using HPLC(C18)-ESI-MS/MS. This sample contained the Fc receptor -chain and the ubiquitous contaminant keratin from human hair and skin cells. Again, CD16a accounted for a majority of the identified peptides.
Surprisingly, human actin and myosin coeluted with CD16a, as well as trace amounts of 97 trypsin and the IgG1 heavy chain. With the exception of the IgG1 heavy chain that leached from the immunoprecipitation column, none of the identified proteins were N- glycoproteins. These results indicate that the PNGase-F treatment of the elution fraction will produce only CD16a N-glycans from NK cells if the immunoprecipitation antibody is treated to remove N-glycans prior to immobilization.
Figure 4.3 (JTR). ESI-MS/MS analysis identifies only CD16a following immunoprecipitation from NK cells. A. Peptides identified are shown with a blue bar under the CD16a sequence. Expected trypsin cleavage sites are indicated with a vertical line and N-glycosylation sites are denoted with a red “N.” B-D. MS/MS spectra of select peptides.
98
N-glycan analysis of CD16a from primary human NK cells
We isolated peripheral NK cells from three male donors ranging in age from 66-
78 to characterize N-glycan composition; blood remaining in the LRS filter after apheresis provided genetic material to determine the donor’s CD16a allotype (Table
4.1, data not shown). Following cell lysis, CD16a immunoprecipitation utilized EndoS- treated 3G8 antibody to eliminate potential contamination with antibody N-glycans (data not shown). Analysis of procainamide-modified CD16a N-glycans by hydrophilic interaction liquid chromatography(HILIC)-ESI-MS showed a strong correlation between
N-glycan elution time and N-glycan mass (Figure 4.4). We identified 33, 27 and 19
CD16a N-glycans from Donors 1, 2 and 3, respectively, with an average mass error of
0.0010 Da for the three datasets (data not shown). The CD16a glycans contained minimally processed forms including a high percentage of oligomannose (23%) and hybrid types (22%; Figure 4.5). Of the remaining complex-type N-glycans (55%), biantennary forms comprised more than half of the complex-type species (51%). CD16a
N-glycan fucosylation proved pervasive with 63% of the total and 89% of the complex- type N-glycans containing this modification (Figure 4.5B). CD16a N-glycans also revealed a high degree of sialylation with 100% of the hybrid and 78% of the complex- type forms displaying at least one N-acetylneuraminic acid residue (Figure 4.5C).
99
Table 4.1. NK cell donor information.
Donor 1 Donor 2 Donor 3
Year of birth 1947 1951 1939
Sex M M M
ABO group A A A
Rh(D) + - +
CD16a allotype V/F V/V V/F
Collection date 5/31/17 5/18/17 5/18/17
NK cell count (106) 206 50 113
NK cell viability 93% 83% 86%
CD56+ in NK cell prep 83.8% 94.1% 95.6%
Analysis of the individual glycoforms corresponding to the ten most intense N- glycan species identified by MS showed complex type, core-fucosylated and sialylated biantennary N-glycans produced the two strongest signals in spectra of CD16a N- glycans from all three donors (Figure 4.6). However, the next eight most intense ion species from each donor were predominantly hybrid forms (13/24) with comparable levels of complex-type (6) and oligomannose (5) forms including Man9 (2), Man 6 (2) and Man5 (1). This prevalence of N-glycan species with restricted processing in the three datasets is surprising given the reports of N-glycans from recombinant CD16a that are primarily of complex type with a high degree of branching (21). 100
Figure 4.4 (JTR). N-glycans identified from Donor 1 NK cell CD16a. The retention time from HILIC for the procainamide-derivatized N-glycans is plotted versus the exact mass. Insets for three N-glycans show MS identification. Cartoon diagrams show one possible configuration of the N-glycan species; isobaric species were not distinguished.
101
Figure 4.5 (JTR). A comparison of CD16a N-glycan characteristics from different sources. A. The pie charts indicate what percentage of identified N-glycans fall into each of the three categories; extracted bar graphs show the breakdown of branching types for the complex-type N-glycans only. B. The percentage of each N-glycan species that contained at least one fucose residue. C. The percentage of each N-glycan species that contained at least one N-acetylneuraminic acid residue.
N-glycan analysis of recombinant CD16a from HEK293 cells
It is possible that CD16a N-glycans isolated from NK cells differ from recombinant CD16a N-glycans because the soluble recombinant fragment lacks the membrane anchor and experiences a different environment during secretion, or that NK cells produce a different mixture of glycan-modifying enzymes, or a combination of both possibilities. We expressed CD16a in HEK293F cells to assess the impact of the transmembrane and intracellular domains on N-glycan composition using two forms: soluble recombinant CD16a that contains the two extracellular antibody-binding domains (srCD16a) and full-length recombinant CD16a containing the complete CD16a 102
ORF, including the signal peptide, coexpressed with the Fc receptor -chain
(frCD16a). It is important to note that recombinant expression of frCD16a requires co- expression with the Fc receptor -chain or CD3 -chain to achieve cell-surface localization ((32) and data not shown).
Migration in SDS-PAGE of srCD16a (28-46 kDa) and frCD16a (38-43 kDa and
47-59 kDa) showed much broader migration distributions than CD16a from NK cells
(53-59 kDa), indicating a greater degree of heterogeneity for the recombinant forms
(data not shown). We identified 122 and 86 individual procainamide-derivatized srCD16a and frCD16a N-glycans, respectively, with an average mass error of 0.0016
Da (data not shown). Predominant N-glycans species from srCD16a and frCD16a were of a complex-type (82% and 73%, respectively) with low levels of oligomannose forms
(5% and 9%, respectively) which stands in marked contrast to NK cell CD16a (Figure
4.5). Furthermore, the complex types showed much greater representation of tri- and tetra-antennary forms than NK cell CD16a. Fucosylation of all srCD16a and frCD16a species was comparable (71%) and both were greater than NK cell CD16a (63%).
Furthermore, fewer srCD16a and frCD16a N-glycans contained N-acetylneuraminic acid when compared to NK cell CD16a. This characterization of srCD16a is highly comparable to expressions in NS0 cells (19) with 6% oligomannose (Man4-6) or BHK cells with no oligomannose (33).
Closer inspection of N-glycan species revealed stark differences between the recombinant forms. The most intense N-glycan signals from srCD16a included a majority of complex-type forms (9/10) in contrast to frCD16a (6/10) that included a significant contribution from oligomannose forms (4/10; including Man9, Man8, Man7 103 and Man5; Figure 4.6). Furthermore, complex-type glycans from frCD16a showed a low level of hexose incorporation (2/6; likely galactose) in contrast to srCD16a with a very high degree of branching (7/9 w/3+ branches) and hexose incorporation (8/9). By these measures, the CD16a membrane anchor and Fc receptor -chain co-expression resulted in the synthesis of a higher degree of hybrid and oligomannose N-glycans, but neither the soluble nor membrane-anchored recombinant CD16a forms recapitulated the N-glycan species found on NK cell CD16a. It is important to note that by using a detergent to solubilize the cell membrane, folded but incompletely-processed CD16a residing in the ER and the Golgi at the moment of cell lysis will be purified in addition to
CD16a from the cell surface. However, frCD16a was extracted using the same technique as NK cell CD16a, thus a direct comparison between these two sources is justifiable. Furthermore, the impact of incompletely-processed CD16a from the ER and
Golgi cannot alone account for the increased prevalence of hybrid forms when compared to frCD16a or srCD16a because 100% of the NK cell CD16a hybrid glycoforms are sialylated, a hallmark of late-stage N-glycan processing.
104
Figure 4.6 (JTR). Possible N-glycan configurations corresponding to the most intense ions are shown from each CD16a source. Cartoon diagrams show one possible organization of the N-glycans; isobaric species were not distinguished. Values in the lower right corner of each cell indicate the relative ion intensities of these species, compared to the most abundant species from the same source.
Oligomannose N-glycans increase receptor affinity
The difference between CD16a N-glycan species from primary NK cells and recombinant expression systems appears compelling because both cell lineage and protein sequence impacted N-glycan remodeling. N-glycan composition can likewise 105 impact protein function, as is the case for IgG1, but it is unknown how composition affects the antibody-binding function of CD16a. To test whether N-glycan composition contributes to CD16a function, we expressed srCD16a in a HEK293S cell line that makes only oligomannose N-glycans with Man5 as the predominant species to compare binding to HEK293F-expressed srCD16a, characterized above, with predominantly complex-type and highly-branched N-glycans (Figures 4.5-4.6) (34).
Figure 4.7 (GPS). CD16a N-glycan composition affect IgG1 Fc binding. Representative SPR sensograms for IgG1 Fc with an agalactosyl core fucosylated complex-type biantennary N-glycan (G0F) binding to recombinant CD16a with complex-type N-glycans (A) or oligomannose N-glycans (B). (C) A summary of dissociation constants for CD16a variants. Material was expressed either with HEK293F cells to synthesize primarily complex-type N-glycans (red bars) or with HEK293S cells to synthesize only oligomannose N-glycans (green bars). Values represent the average of at least two independent experiments and error bars are +/- the maximum error of the fitting.
106
CD16a with complex-type N-glycans bound IgG1 Fc in the G0F glycoform with a
KD of 310 ±100 nM and 12-fold less affinity than CD16a with oligomannose N-glycans
(25 ±17 nM; Figure 4.7). Furthermore, two of the five CD16a N-glycans previously identified as positive contributors to antibody binding, N45 and N162 (35-38), but not the remaining three N-glycans at positions 38, 74 and 169, were required for the enhanced antibody binding by CD16a displaying oligomannose N-glycans. Removing CD16a N- glycans at positions 45 and 162 by N to Q mutations abolished enhancement from the oligomannose N-glycans. These data indicate that composition of the N45 and N162
CD16a N-glycans impact function.
NMR of different srCD16a N-glycoforms
Our laboratory previously observed that different IgG1 Fc N-glycoforms led to different CD16a binding. These changes correlated with differences in crosspeak positions observed with solution nuclear magnetic resonance (NMR) spectroscopy and these differences reflected changes in secondary structure for one IgG1 Fc loop (39).
We applied a similar approach and incorporated selective backbone 15N labels into Y, K and F residues to probe whether N-glycan composition affected 1H-15N heteronuclear single quantum coherence (HSQC) spectra of srCD16a. The 26 Y, F and K residues are evenly distributed throughout both CD16a extracellular domains. We expect changes in peak positions for these amino acid types will reflect CD16a structural changes in a manner analogous to characterizing ligand binding through a NMR crosspeaks that change position during a titration. Indeed, a spectrum of srCD16a expressed in
HEK293S cells with oligomannose N-glycans showed different cross peak positions 107 than srCD16a with complex-type N-glycans (Figure 4.8). The largest deviations appeared in the center of the spectrum. We observed 24 peaks which is close to the 26 expected, and of these, 8 showed different crosspeak positions in the two spectra with a combined 1H/15N chemical shift perturbation for each peak > 0.04 ppm as calculated with Eq 2. Furthermore, one peak present in the spectrum of CD16a with complex-type
N-glycans was absent in the spectrum of CD16a with oligomannose N-glycans and two peaks appeared in the spectrum of the oligomannose form. It is important to note that only two of the 26 labeled residues are within five amino acid residues, by primary sequence, from N-glycosylation sites indicating extensive regions of the protein are affected by N-glycan composition. This result is substantially different from hIgG1 Fc, where changes are limited to a single loop and not observed throughout the protein
(39).
Figure 4.8 (GPS). CD16a N-glycan composition impacts polypeptide structure. Select backbone amide crosspeaks observed in a HSQC experiment using CD16a expressed with either complex-type (red contours) or oligomannose (black contours) N-glycans. One peak present in the spectrum of CD16a with complex-type N-glycans but absent in the oligomannose form is indicated (†). Two peaks appearing in the spectrum with oligomannose N-glycans are indicated (*). 108
Discussion
A high percentage of oligomannose and hybrid structures, as well as biantennary forms constituting the largest subgroup of the complex-type species, characterize the
CD16a N-glycans from primary human NK cells and indicate restricted processing compared to many serum proteins. This limited processing of NK cell CD16a N-glycans stands in stark contrast to N-glycans from recombinant CD16a with highly-modified and highly-branched carbohydrate structures. Furthermore, we provide clear evidence that
N-glycan composition impacts antibody binding: CD16a modified with minimally processed oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than
CD16a with a mixture of highly-processed N-glycans.
Recombinant CD16a is often used to characterize antibody binding affinity in many reports, including some of our own, due to practical considerations that include isolating sufficient material for in vitro studies. The differences in N-glycan composition from these different sources highlight the need to describe and develop the tools to mimic native source-specific composition in studies of receptor function. N-glycan composition is a neglected component of eukaryotic biology, particularly in the context of cell surface proteins, because access to primary materials is limited and the absolute mass of each specific receptor on a cell surface is very low (we estimate ~20-40 fg
CD16a / NK cell). Furthermore, detailed studies linking N-glycan composition with function are challenging and likewise represent a barrier to a complete characterization of N-glycan roles in protein function. The characterization presented here provides a detailed description of these variables for CD16a from NK cells. N-glycosylation of 109
CD16a was targeted due to its relevance in mAb therapy but the other Fc receptors are heavily glycosylated. This approach can be used to characterize N-glycosylation profiles of other Fc receptors from primary human tissues.
The relatively limited distribution of migration rates for NK cell CD16a in polyacrylamide gels and bias towards N-glycoforms with restricted processing was striking in comparison to frCD16a from HEK293F cells and indicates cell-type specific differences in N-glycan processing. Both frCD16a and NK cell CD16a were co- expressed with a co-receptor that is required for localization to the cell surface.
Differences in primary structure do not account for the observed differences in N-glycan processing: the CD16a polypeptide sequence and presence of the co-expressed Fc receptor -chain were the same in both NK cells and that used in HEK293F cells to express frCD16a. Cell-type specific differences in the glycosylation of a specific protein are known (40-44) and are justifiable given the complexity of gene expression and activity for glycan-processing enzymes in different tissues (45-50).
Changes in receptor function resulting from limited N-glycan processing were mirrored by changes in a 2d spectrum of [15N-Lys/Tyr/Phe]-labeled CD16a (Figures
4.7-4.8). The magnitude of peak shifts and the extent of peaks experiencing shifts in
CD16a are comparable to analyses of proteins that experience a conformational rearrangement upon ligand binding. In the latter example, ligand docking stabilizes a specific protein conformation. Surprisingly, CD16a N-glycan composition imposed a similar conformational adjustment that was experienced by a large number of backbone amides, indicating that specific features of the N-glycans interact with residues on the polypeptide surface. Our group recently described the formation of intramolecular 110 interactions between the polypeptide and N-glycans at N45 and N162 (38). N-glycans at these positions likely differentially impact protein conformation, based on N-glycan composition at each site. Thus, the CD16a N-glycans do not behave as largely inert modifications, but rather interact with polypeptide residues to modulate protein function.
The IgG1 Fc N-glycan composition also impacts receptor binding through intramolecular interactions (15,39,51,52).
Numerous prior studies focused on the modulation of CD16a affinity by IgG N- glycan composition. This wealth of information on IgG N-glycosylation is due primarily to the availability of IgG; here we focused on the effect of CD16a N-glycosylation on antibody binding. The observation that CD16a N-glycosylation affects binding affinity, potentially to a greater extent than IgG, makes CD16a N-glycosylation an important aspect of immune system function with the potential for clinical implications. There are two important limitations to the present study: 1) the restricted donor demographic variability could mask potential N-glycan composition variability present in the larger population and 2) it is currently unclear if changes to CD16a N-glycan composition that increase antibody binding in SPR experiments enhance NK cell response. Moreover,
CD16a-expressing cells, including NK cells, are involved with multiple pathologies (53-
56). Therefore, this information regarding specific NK cell N-glycans on CD16a can be applied to probe the connection between patient-specific changes in autoimmune conditions, malignancy or infection.
111
Experimental Procedures
All materials purchased from Sigma-Aldrich unless noted.
Cell isolation
NK cells were isolated from leukocyte reduction filters (LRS filters) obtained from
LifeServe (Ames, IA) and DeGowin Blood Centers (Iowa City, IA) after the plateletpheresis procedure. Donors signed consent forms permitting the use of donated blood products for research purposes. We do not directly enroll donors because we obtain materials from the LifeServe and DeGowin Blood centers. Cell isolation procedures were performed within 3 h of the time the donor completed the apheresis procedure. NK cells were isolated by negative selection using the RosetteSep
Enrichment Cocktail following the recommended protocol (Stem Cell Tech.) with the following minor changes: LRS filters were drained into a sterile 50 mL tube and the contents diluted to 30 mL using PBS with 2% IgG-depleted FBS. The cell suspension was then split into 2 x15 mL aliquots and each mixed with 750 µl of the negative selection cocktail. After a 20 min incubation, the steps mentioned in the protocol were followed. Contaminating erythrocytes were then removed by incubating the cells with erythrocyte lysis buffer (155 mM ammonium chloride, 12 mM sodium bicarbonate, and
0.1 mM ethylenediamenetetraacetic acid, pH 7.4) for 10 min at RT. Finally, cells were counted and viability analyzed with trypan blue, then frozen at -80 °C. CD16a allotype was determined by sequencing cDNA corresponding to CD16a (cDNA isolation described below in the recombinant expression section; ISU DNA facility).
112
Flow cytometry
NK cells were first blocked with 5 µg hIgG1 (Athens Research and Technology) before incubation with primary antibodies. For each flow cytometry experiment, 5 x 105
NK cells were stained with primary antibody for 40 min in dark on ice, including 2 µg/mL mIgG2a anti-hCD56 (MEM-188, BioLegend) and 2 µg/mL mIgG1 anti-hCD16 (3G8) or isotype controls, mIgG1 (Research and Diagnostics Systems) and mIgG2a
(BioLegend). The cells were washed with 2 x 2 mL phosphate buffered saline, 0.05% sodium azide by centrifuging at 600 xg for 8 min and removing the supernatant after each centrifugation. Fluorophore-conjugated secondary antibodies were added including anti mIgG1-APC (RMG1-1, BioLegend) and anti mIgG2a-PE (RMG2a-62,
BioLegend) and incubated on ice for 40 min. Cells were fixed in 1% paraformaldehyde before loading them onto a BD FACSCanto (BD Biosciences). For cell purity assessment, NK cells were gated in the side and forward scatter plot to exclude cell debris and cells (primarily erythrocytes) smaller than lymphocytes. Gating of double stained cells was determined by comparing to the fluorescence intensity of the negative
(no primary) control. Isotype controls consistently showed no positive staining for either primary antibody for the first 15 NK cell isolations performed from donors of both gender and a wide age range.
Anti-hCD16 expression and purification
Open reading frames encoding the anti-hCD16 mouse IgG1 (3G8) heavy and light chains were synthesized (IDT). The heavy chain sequence was cloned into pGEef1Puro vector (provided by Dr. Kelley Moremen, University of Georgia) using the 113
Gateway cloning system (Life Tech). The flanking attB sites for gateway cloning of heavy chain were included in the synthesized heavy chain gene. The transfer of gene to final pGEef1Puro vector was performed in a two-step gateway reaction following manufacture’s protocol with pDONR221 (Life Tech) as the intermediate vector. The light chain sequence was cloned into the pGEN2 vector using the NotI and HindIII restriction sites (57).
3G8 monoclonal antibody was generated by co-transfecting HEK293F cells with
3 g/mL pGEef1Puro:(heavy chain) and 1.5 g/mL pGEN2:(light chain) as described
(57). Antibody was secreted into the medium, adsorbed to a protein-A Sepharose fast flow resin (GE Life Sciences) and eluted with 100 mM glycine, pH 3.0 followed by immediate neutralization with 33 % 1 M Tris, pH 8.0. Purified antibody was washed to replace the buffer with a buffer containing 25 mM MOPS, 100 mM sodium chloride, pH.7.2 and then coupled to AminoLink aldehyde-functionalized agarose beads at 1 mg/mL of resin according to the product protocol (Thermo Scientific).
Antibody used to immunoprecipitate CD16a for N-glycan analysis differed in two important ways. First, the mouse IgG1 Fc region was replaced with human IgG1 Fc.
Second, this chimeric antibody was treated with EndoS to remove the Fc N-glycan; 3G8 contains no N-glycosylation sequon in the Fab domains. EndoS was expressed in E. coli using a deposited plasmid and purified as described (AddGene). EndoS was incubated with 3G8 in 20 mM MOPS, 100 mM NaCl, pH 7.2, at a 1:50 (EndoS:3G8) molar ratio for 18 h at RT. The digestion was assessed by resolving the protein with
SDS-PAGE and staining with coomassie blue. Glutathione beads were incubated with 114
3G8 and GST-EndoS to remove the GST-EndoS before coupling antibody to agarose beads.
Recombinant CD16a expression
The soluble extracellular fragment of CD16a (srCD16a) was prepared as a recombinant GFP-fusion protein and cleaved with TEV as previously described using
HEK293F cells (15). Open reading frames encoding the full-length recombinant CD16a protein (frCD16a) and the Fc receptor subunit (-chain) were cloned from cDNA obtained from NK cells and monocytes, respectively. Total RNA was isolated from
10x106 monocytes (prepared with negative selection, Stem Cell Tech) and 5x106 NK cells using the Trizol reagent following the manufacturer’s protocol (ThermoFisher). A high capacity RNA to cDNA kit (Applied Biosystems) was used to reverse transcribe
500 ng RNA from each cell type and amplified with AccuPrime Pfx DNA polymerase
(Life Tech) to generate full length cDNA. Primers pairs for frCD16a were: 5'- cagcggccgccCAGTGTGGCATCATGTGGCAG and 5'- ggatccaagcttTTTGTCTTGAGGGTCCTTTCT containing NotI and HindIII sites, respectively. Similarly, two primers 5'-gaattcCTCCAGCCCAAGATGATTCCAG and 5'- ggatcctcaCTACTGTGGTGGTTTCTCATGC with EcoRI and BamHI sites, respectively, were used to amplify gene for -chain. Primers were designed to include the native signal sequence form both genes. Both PCR fragments were cloned into pGEM-T Easy vectors following the manufacturer’s guidelines (Promega). Full-length CD16a was sub- cloned into pGEN2 vector using the NotI and HindIII restriction sites. The -chain was sub-cloned into pGEef1Puro vector using the EcoRI and BamHI restriction sites. 115 pGEN2-frCD16a and pGEef1Puro-γ-chain vector were co-transfected in a 1:2 mass ratio in HEK293F cell line as described (57). HEK cells were transfected at a density of
3.0 x106 cells mL-1 with a viability >96%. Expressions usually proceeded for 5 d or until cell viability fell below 50%. Typical yields for srCD16a were 0.2 mg protein mL-1 culture medium. Cell surface expression of CD16a in HEK293F cells was confirmed by flow cytometry using the anti-CD16 mIgG1 (3G8) antibody before freezing the cell pellets at -
80°C
CD16a Immunoprecipitation
These methods were adapted from a previously published protocol to achieve a preparative scale purification (58). NK cell or HEK293F cells expressing full-length
CD16a were lysed by resuspending the frozen pellet in ice-cold lysis buffer (100 mM
Tris, 100 mM sodium chloride, 5 mM ethylenediamenetetraacetic acid, 5 mM oxidized glutathione, 10 µM potassium ferricyanide, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 mg/mL dodecylmaltoside, pH 8.0) with 200 µL added for every 1x107 cells by repeated pipetting, followed by a 20 min incubation on ice. Cell debris was removed by centrifugation at 10,000 xg for 10 min at 4 °C. Supernatant was incubated for 1 hr. at
4 °C on a rocker with 5 µL protein G sepharose (Sigma Aldrich) per 200 µl lysate.
Protein G sepharose was removed by centrifuging the lysate at 500 xg for 5 min. The recovered supernatant was sonicated in a bath sonicator for 1 min, followed by freezing at -80 °C and slowly thawed on ice. 3G8-coupled agarose beads (20 µL) were added per 200 µL NK cell lysate and incubated for 1 h at 4 °C on a rocking shaker. The resin was centrifuged at 500 xg for 5 min and washed twice with lysis buffer using 10x the 116 resin volume for each wash followed by two washes with 50 mM Tris, 100 mM sodium chloride, pH 8.0 and final two washes with 100 mM ammonium carbonate, pH 8.0.
CD16 was eluted in 45:55:0.1 (HOH:acetonitrile:TFA) by centrifugation through a spin column to retain the resin.
PNGase F digestion of CD16a
PNGase F digestion of CD16a isolated from NK cells was performed per manufacturer’s protocol. NK cell CD16a bound to 3G8-agarose was boiled at 100 °C for
10 min in 1x denaturing buffer (0.5% sodium dodecylsulphate, 40 mM dithiothreitol) followed by addition of GlycoBuffer2 and 10% NP-40 to the reaction (New England
Biolabs). After adding 1 µL PNGase F, the sample was incubated at 37 °C for 4 h. and the reaction was stopped by heating at 95 °C for 5 min after the addition of 1 volume 2x
SDS-PAGE sample buffer containing 10% beta-mercaptoethanol. A control reaction with CD16a was treated in an identical manner, except without the addition of PNGase
F.
Western blotting
Eluted protein was resolved on a sodium dodecyl sulfate polyacrylamide gel
(12% unless mentioned otherwise) and transferred onto a Polyvinylidene difluoride
(PVDF) membrane using the one-step electroblotting system (ThermoFisher). The membrane was blocked with 5% dry milk in TBS Tween-20 (TBST) buffer for 1 h. at RT.
All incubations and washes were performed on an orbital shaker. Following blocking, the membrane was stained for 18 h at 4 °C with anti-hCD16 (Research and Diagnostic 117
Systems, 0.1 µg/ml) in 5% milk in TBST buffer. The blot was washed 4x with TBST buffer for 5 min and stained with secondary anti-goat-HRP (Research and Diagnostic
Systems, dilution 1:2000) in 5% milk in TBST buffer for 1 h at RT. After 4x washing the blot was imaged on a ChemiDoc XRS+ Image system (BioRad) using the ECL Western
Blotting Substrate (ThermoFisher).
Peptide mass spectrometry
CD16 for peptide sequencing was isolated as described in the immunoprecipitation protocol. In the first instance, CD16a was excised from 12% SDS-
PAGE at the same size as detected by the western blot detection system. The gel was subjected to standard in-gel trypsin digest on the Molecular Dynamics ProGest
(Genomic Solutions) instrument. The sample was then dried down and resuspended in
1.25 µL buffer B (0.1% formic acid in acetonitrile). In the second instance, peptides retained by the C18 sep-pak during the isolation of N-glycans from donor 3 (following treatment with PNGaseF and trypsin) were eluted with isopropanol and lyophilized. Both samples were resuspdended in 23.75 µL buffer A (0.1% formic acid in water) was added to the sample, mixed, spun down and put in a sample vial. The vial was then loaded onto a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo
Scientific) equipped with an 1260 HPLC (Agilent) and C18 column and eluted as described (59). Peaks from MS1 and MS2 spectra were searched with the mammalian
Mascot database server. Raw data were processed with Sequest HT (v 1.17) and
Mascot 2.2.07 (Matrix Science). The peptides identified were then analyzed using
Proteome Discoverer Version 2.1 software (ThermoFisher). 118
Procainamide labeling of PNGase F-released N-glycans
Following heat denaturation of CD16a for 5 min at 90 °C, 1 µL sequencing grade trypsin (Promega; 1 mg/mL in 50 mM ammonium carbonate, pH 8.0) was added and incubated at 37 °C for 18 h after mixing. The trypsinized sample was again incubated at
90 °C for 5 min to denature the trypsin, followed by cooling for 5 min on ice. PNGase F
(1 µL; glycerol-free; New England BioLabs) was then added to the sample and incubated at 37 °C for 18 h. A Hypersep C18 column (ThermoFisher) was washed 3x1 mL of methanol then 3x1 mL 5% acetic acid. Free N-glycans were isolated by applying the sample, diluted in 5% acetic acid, over the C18 column and collecting the flow through.
Wash fractions (3x1 mL) of 5% acetic acid were collected in the same tube, then frozen and lyophilized. The lyophilized sample was then resuspended in 200 µL of double- distilled water by vortexing and transferred to a microcentrifuge tube, frozen and lyophilized again. The sample was then resuspended in 10 µL of water. A stock solution containing 2 M sodium cyanoborohydride, 1 M procainamide and 50% acetic acid (10 µL) was added to the sample, mixed and incubated for 18 h at 37 °C. The sample was then lyophilized and resuspended into 10 µL of double-distilled water.
Hydrophilic interacting chromatography mass spectrometry
The HPLC instrument used was an Agilent 1260 HPLC with an Acquity UPLC
Glycoprotein BEH Amide, 300 Å, 1.7 µm Hydrophilic Interaction Liquid Chromatography
(HILIC) column (Waters). The lyophilized procainamide-labeled glycan samples were resuspended in double-distilled water and 9 µL was injected onto the column 119 prewashed with 25% Solvent A (0.1% formic acid and 0.01% TFA in double-distilled water) and 75% Solvent B (0.1% formic acid and 0.01% TFA in acetonitrile) with a flow rate of 0.1 mL/min. The glycans were then eluted by a gradient at 0.1 mL/min flow rate unless otherwise indicated; 0.5-10.5 minutes (gradient from 25%-35% A), 10.5-40.5 minutes (a gradient from to 35% to 50% A), 40.5-41.5 minutes (50% to 100% A), 41.5-
43.5 minutes (100% A), 46.5-47 minutes (25% A, 75% B) at 0.05 mL/min and 47-60 minutes (25% A, 75% B) at 0.1 mL/min at 45 °C. Upon elution, the samples were coupled in-line to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer
(Thermo Scientific). The mass spectrometer (scan range 266.7- 4000 m/z) subjected ions to HCD fragmentation (scan range of 200-2000 m/z) at a normalized collisional energy of 27 eV. In between samples, identical chromatography and MS analyses were performed following an injection of water. These were scanned to ensure no carryover between samples. Sample spectra were then analyzed by converting the RAW spectra file to text format with MSConvert. The output was then scanned against a list of N- glycans calculated for singly, doubly and triply charged, procainamide-derivatized species to identify peaks with corresponding retention times and intensities. These matches were then validated manually by analyzing the RAW file in XCaliber (Thermo
Scientific). Average mass error for each data set was calculated according to equation
1.
∑푛|푚/푧푐푎푙푐− 푚/푧표푏푠| 푒푟푟표푟 = 1 푛 푛 Eq. 1 푛
120
IgG1 Fc binding assays by SPR
Expression, purification and preparation of IgG1 Fc in the G0F glycoform and
CD16a binding analyses by surface plasmon resonance were performed as described
(52). Dissociation constants were fitted to the equilibrium response units for each condition.
NMR spectroscopy
Isotope-labeled srCD16a expression and NMR experiments were conducted as described (39). Chemical shift perturbations (CSP) were calculated using equation 2.
1 퐶푆푃 = √(훿 1퐻)2 + (훿15푁)2 Eq. 2 6
Acknowledgement
We thank Christine Hayes and the staff of the LifeServe Blood Center (Ames and
Des Moines, IA) and the staff of the DeGowin Blood Center (U. of Iowa Medical Center) for the Trima LRS filters, Dr. William Alley (Waters) for help with the HILIC separations,
Prof. Kelley Moremen (UGA) for the pGEef1 vector, Dr. Shawn Rigby of the ISU Flow
Cytometry Facility for flow cytometry analysis and Mr. Joel Nott of the ISU Protein
Facility for mass spectrometric analyses. This material is based upon work supported by the National Institutes of Health under Award No. R01 GM115489 (NIGMS), and by funds from the Roy J. Carver Department of Biochemistry, Biophysics & Molecular
Biology at Iowa State University. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institutes of Health. 121
Conflict of Interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author Contributions
KRP, JTR, GPS and AWB designed experiments, analyzed the results, wrote and approved the final version of the manuscript. KRP and JTR purified cells and analyzed N- glycans from recombinant and primary sources. GPS analyzed in vitro binding and acquired NMR spectra.
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21. Hayes, J. M., Frostell, A., Karlsson, R., Muller, S., Martin, S. M., Pauers, M., Reuss, F., Cosgrave, E. F., Anneren, C., Davey, G. P., and Rudd, P. M. (2017) Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab. Mol Cell Proteomics 16, 1770-1788 22. Taniguchi, T., Woodward, A. M., Magnelli, P., McColgan, N. M., Lehoux, S., Jacobo, S. M. P., Mauris, J., and Argueso, P. (2017) N-Glycosylation affects the stability and barrier function of the MUC16 mucin. J Biol Chem 292, 11079-11090 23. Higel, F., Seidl, A., Sorgel, F., and Friess, W. (2016) N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins. Eur J Pharm Biopharm 100, 94-100 24. Nagae, M., and Yamaguchi, Y. (2012) Function and 3D structure of the N-glycans on glycoproteins. Int J Mol Sci 13, 8398-8429 25. Passlick, B., Flieger, D., and Ziegler-Heitbrock, H. W. (1989) Identification and characterization of a novel monocyte subpopulation in human peripheral blood. Blood 74, 2527-2534 26. Neron, S., Thibault, L., Dussault, N., Cote, G., Ducas, E., Pineault, N., and Roy, A. (2007) Characterization of mononuclear cells remaining in the leukoreduction system chambers of apheresis instruments after routine platelet collection: a new source of viable human blood cells. Transfusion 47, 1042-1049 27. Lanier, L. L., Ruitenberg, J. J., and Phillips, J. H. (1988) Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes. J Immunol 141, 3478-3485 28. Edberg, J. C., Redecha, P. B., Salmon, J. E., and Kimberly, R. P. (1989) Human Fc gamma RIII (CD16). Isoforms with distinct allelic expression, extracellular domains, and membrane linkages on polymorphonuclear and natural killer cells. J Immunol 143, 1642-1649 29. Ravetch, J. V., and Perussia, B. (1989) Alternative membrane forms of Fc gamma RIII(CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions. J Exp Med 170, 481-497 30. Edberg, J. C., Barinsky, M., Redecha, P. B., Salmon, J. E., and Kimberly, R. P. (1990) Fc gamma RIII expressed on cultured monocytes is a N-glycosylated transmembrane protein distinct from Fc gamma RIII expressed on natural killer cells. J Immunol 144, 4729-4734 31. Fleit, H. B., Wright, S. D., and Unkeless, J. C. (1982) Human neutrophil Fc gamma receptor distribution and structure. Proc Natl Acad Sci U S A 79, 3275-3279 32. Blazquez-Moreno, A., Park, S., Im, W., Call, M. J., Call, M. E., and Reyburn, H. T. (2017) Transmembrane features governing Fc receptor CD16A assembly with CD16A signaling adaptor molecules. Proc Natl Acad Sci U S A 114, E5645-E5654 124
33. Takahashi, N., Cohen-Solal, J., Galinha, A., Fridman, W. H., Sautes-Fridman, C., and Kato, K. (2002) N-glycosylation profile of recombinant human soluble Fcgamma receptor III. Glycobiology 12, 507-515 34. Reeves, P. J., Callewaert, N., Contreras, R., and Khorana, H. G. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N- acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99, 13419-13424 35. Ferrara, C., Stuart, F., Sondermann, P., Brunker, P., and Umana, P. (2006) The carbohydrate at FcgammaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms. J Biol Chem 281, 5032-5036 36. Ferrara, C., Grau, S., Jäger, C., Sondermann, P., Brünker, P., Waldhauer, I., Hennig, M., Ruf, A., Rufer, A. C., Stihle, M., Umaña, P., and Benz, J. (2011) Unique carbohydrate–carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose. in Proc Natl Acad Sci U S A. pp 12669-12674 37. Mizushima, T., Yagi, H., Takemoto, E., Shibata-Koyama, M., Isoda, Y., Iida, S., Masuda, K., Satoh, M., and Kato, K. (2011) Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans. Genes Cells 16, 1071-1080 38. Subedi, G. P., Falconer, D. J., and Barb, A. W. (2017) Carbohydrate-Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy. Biochemistry 56, 3174-3177 39. Subedi, G. P., and Barb, A. W. (2015) The Structural Role of Antibody N- Glycosylation in Receptor Interactions. Structure 23, 1573-1583 40. Liu, Y., Chen, J., Sethi, A., Li, Q. K., Chen, L., Collins, B., Gillet, L. C., Wollscheid, B., Zhang, H., and Aebersold, R. (2014) Glycoproteomic analysis of prostate cancer tissues by SWATH mass spectrometry discovers N-acylethanolamine acid amidase and protein tyrosine kinase 7 as signatures for tumor aggressiveness. Mol Cell Proteomics 13, 1753-1768 41. West, M. B., Segu, Z. M., Feasley, C. L., Kang, P., Klouckova, I., Li, C., Novotny, M. V., West, C. M., Mechref, Y., and Hanigan, M. H. (2010) Analysis of site-specific glycosylation of renal and hepatic gamma-glutamyl transpeptidase from normal human tissue. J Biol Chem 285, 29511-29524 42. Medzihradszky, K. F., Kaasik, K., and Chalkley, R. J. (2015) Tissue-Specific Glycosylation at the Glycopeptide Level. Mol Cell Proteomics 14, 2103-2110 43. Sethi, M. K., Kim, H., Park, C. K., Baker, M. S., Paik, Y. K., Packer, N. H., Hancock, W. S., Fanayan, S., and Thaysen-Andersen, M. (2015) In-depth N-glycome profiling of paired colorectal cancer and non-tumorigenic tissues reveals cancer-, stage- and EGFR- specific protein N-glycosylation. Glycobiology 25, 1064-1078 44. Loke, I., Ostergaard, O., Heegaard, N. H. H., Packer, N. H., and Thaysen- Andersen, M. (2017) Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated 125
Human Neutrophil Elastase Modulates Its Immune Functions. Mol Cell Proteomics 16, 1507-1527 45. Yamamoto, M., Yamamoto, F., Luong, T. T., Williams, T., Kominato, Y., and Yamamoto, F. (2003) Expression profiling of 68 glycosyltransferase genes in 27 different human tissues by the systematic multiplex reverse transcription-polymerase chain reaction method revealed clustering of sexually related tissues in hierarchical clustering algorithm analysis. Electrophoresis 24, 2295-2307 46. Nairn, A. V., York, W. S., Harris, K., Hall, E. M., Pierce, J. M., and Moremen, K. W. (2008) Regulation of glycan structures in animal tissues: transcript profiling of glycan- related genes. J Biol Chem 283, 17298-17313 47. Kaburagi, T., Kizuka, Y., Kitazume, S., and Taniguchi, N. (2017) The Inhibitory Role of alpha2,6-Sialylation in Adipogenesis. J Biol Chem 292, 2278-2286 48. Wang, Y. C., Stein, J. W., Lynch, C. L., Tran, H. T., Lee, C. Y., Coleman, R., Hatch, A., Antontsev, V. G., Chy, H. S., O'Brien, C. M., Murthy, S. K., Laslett, A. L., Peterson, S. E., and Loring, J. F. (2015) Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells. Sci Rep 5, 13317 49. Huang, H. H., and Stanley, P. (2010) A testis-specific regulator of complex and hybrid N-glycan synthesis. J Cell Biol 190, 893-910 50.Huang, H. H., Hassinen, A., Sundaram, S., Spiess, A. N., Kellokumpu, S., and Stanley, P. (2015) GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain. Elife 4 51. Barb, A. W. (2015) Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1. Biochemistry 54, 313-322 52. Subedi, G. P., and Barb, A. W. (2016) The immunoglobulin G1 N-glycan composition affects binding to each low affinity Fc gamma receptor. MAbs 8, 1512-1524 53. Dalbeth, N., and Callan, M. F. (2002) A subset of natural killer cells is greatly expanded within inflamed joints. Arthritis Rheum 46, 1763-1772 54. Jonsen, A., Gunnarsson, I., Gullstrand, B., Svenungsson, E., Bengtsson, A. A., Nived, O., Lundberg, I. E., Truedsson, L., and Sturfelt, G. (2007) Association between SLE nephritis and polymorphic variants of the CRP and FcgammaRIIIa genes. Rheumatology (Oxford) 46, 1417-1421 55. Izumi, Y., Ida, H., Huang, M., Iwanaga, N., Tanaka, F., Aratake, K., Arima, K., Tamai, M., Kamachi, M., Nakamura, H., Origuchi, T., Kawakami, A., Anderson, P., and Eguchi, K. (2006) Characterization of peripheral natural killer cells in primary Sjogren's syndrome: impaired NK cell activity and low NK cell number. J Lab Clin Med 147, 242- 249 56. Schepis, D., Gunnarsson, I., Eloranta, M. L., Lampa, J., Jacobson, S. H., Karre, K., and Berg, L. (2009) Increased proportion of CD56bright natural killer cells in active and inactive systemic lupus erythematosus. Immunology 126, 140-146 126
57. Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. W., and Barb, A. (2015) High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. J Vis Exp, e53568 58. Edberg, J. C., Barinsky, M., Redecha, P. B., Salmon, J. E., and Kimberly, R. P. (1990) Fc gamma RIII expressed on cultured monocytes is a N-glycosylated transmembrane protein distinct from Fc gamma RIII expressed on natural killer cells. J Immunol 144, 4729-4734 59. Marcella, A. M., and Barb, A. W. (2017) The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins). Appl Microbiol Biotechnol
127
CHAPTER 5. GENERAL CONCLUSION
The interaction between IgG1 and FcRs is modulated by N-glycosylation composition. CD16a and CD16b are nearly identical FcRs which bind to IgG1 with different affinities. The role of a single amino acid residue, G129 in CD16a or D129 in
CD16a, was explained to be the major difference in binding between these two receptors using SPR. Furthermore, the first crystal structure of glycosylated CD16b in complex with IgG1 Fc provided important structural information which explain the energetic penalty of residue D129 in CD16b as compared to G129 in CD16a. CD16 with
G129 was also described to have enhanced affinity for IgG1 when expressing Man5 N- glycans on the surface rather than complex-type. These studies may provide essential information which help optimization of mAb therapies for the purposes of selectively targeting CD16a or CD16b.
Endogenous FcR N-glycosylation was previously undiscovered before this work.
Our previous in vitro studies indicate CD16a N-glycan composition affects binding to
IgG1, thus it was important to understand what compositions are found in vivo. These studies indicate CD16a and CD32a N-glycosylation from monocytes vary by site, donor and cell type. The current population size is quite limited at this point, but methods developed can now be applied to a larger population size to understand N-glycan processing differences from individuals who vary by gender, age, and disease states. 128
APPENDIX A. A RARE ALLOTYPE ON CD16A FROM MONOCYTES ISOLATED FROM PLATELET APHERESIS DONORS AFFECT N-GLYCOSYLATION COMPOSITION AT SITE N45
Introduction
CD16a is predominantly expressed on NK cells and a subset of monocytes.
CD16a is the main receptor for antibody dependent cellular cytotoxicity (ADCC) in NK cells and nonclassical (CD14++, CD16+) monocytes (1). CD16a is N-glycosylated at five N-glycosylation sites; N38, N45, N74, N162 and N169. CD16a N-glycosylation and polymorphisms have been described to affect binding to IgG antibodies (2-3). Two main polymorphisms exist on CD16a which dictate binding and function; L/H/R48 and
V/F158. Single copy allele frequencies for residue 48 are estimated to be 84% L48, 8%
H48 and 6% R48 (7). Rare homozygous individuals for the H48 allele suffer from severe recurrent herpes virus infections as well as frequent upper respiratory infections (4-8).
Interestingly, NK cell ADCC was unaffected but H48-encoding homozygotes did have compromised natural cytotoxicity (4-5). This finding suggests the H48 polymorphism does not affect IgG binding to CD16a unlike the V/F158 polymorphism. CD16a V158 is associated with higher binding to antibody than the F158 allele and in vivo V158 homozygous individuals respond better to mAb therapies presumably because of this higher affinity (9-10). The CD16a H48 allele is described to abrogate the interaction between CD16a and CD2 shown through immunoprecipitation and confocal microscopy co-localization experiments implicating CD16a function as a co-stimulatory receptor for natural cytotoxicity along with its canonical role in ADCC (11). Patients homozygous for
H48 in this study were described to have decreased CD2 expression as well. CD16a 129
H48 has been relatively well studied in the context of NK cells but has not been studied in the context of nonclassical monocytes nor has the role of N-glycosylation of CD16a from individuals with the H48 allele been elucidated before. CD16a N-glycosylation has recently been characterized from primary tissue including monocytes (Chapter 3) and
NK cells (12). In this study, N-glycosylation compositions are compared between two allele-specific CD16a N45 N-glycopeptides containing the L48 or H48 residue from human monocytes.
Results
The CD16a allotype H48 causes different N-glycosylation processing in monocytes at site N45 as compared to L48. The CD16a H48 specific allele was found on the N45 glycopeptide in the dataset including a pool of 3 donors (MoB) as described in Chapter 3. MS2 fragmentation confirmed these allele specific glycopeptides. The diagnostic peptide + GlcNac ion was observed for the L48 (Figure A.1A) and H48 alleles (Figure A.1D-E). These peptides are the product of a missed Glu-C cleavage site while the fully cleaved product is displayed in Figure A.1B and A.1C. MoB had a single donor, Mo06iii, which was confirmed by DNA sequencing to be heterozygous for both the V/F158 and L/H48 alleles (Figure 3.1F) (Chapter 3). The relative intensities for all monocyte donors with the L48 glycopeptide (peptide 2) and the H48 glycopeptides were analyzed for site N45 (Figure A.2). The 10 most abundant N-glycans from all monocytes at site N45 from peptide 2 are summarized in Figure A.2A for the L48 allele.
These data are from the same donors analyzed in Chapter 3. Six N-glycans identified on the CD16a N45 L48 glycopeptide were hybrid type while there were two complex and two oligomannose type in the 10 most intense glycopeptides. Percent composition 130 of the top 10 N-glycans from peptide 2 was 68% hybrid, 23% complex and 9% oligomannose-type N-glycans. Figure A.2B displays the top 10 most abundant N- glycans identified from the CD16a N45 H48-specific glycopeptide. Interestingly, there were 5 hybrid, 5 complex and no oligomannose types identified and percent composition was 62% hybrid and 38% complex-type suggesting greater processing of the N45 N-glycan when H48 is present.
Figure A.1. MS2 fragmentation and DNA sequencing confirms L/H48 specific glycopeptides from a monocyte donor pool. A) CD16a N45 L48 allele-specific hybrid-type glycoepeptide is a missed Glu-C cleavage site product. B-C) CD16a N45 oligomannose and hybrid type glycopeptides with Glu-C cleavage does not allow the L/H allele peptide to be distinguished. D-E) CD16a N45 H48 allele-specific hybrid-type glycopeptides are missed Glu-C cleavage site products. F) Sanger sequencing confirms this donor to be heterozygous for V/F158 and L/H48 alleles. A-E) Oxonium ions for mono, di and trisaccharides are identified and labeled appropriately. The peptide + GlcNac peak is labeled appropriately and confirms peptide-specific glycopeptides. 131
Figure A.2. The top 10 N-glycans identified on CD16a N45 is shifted to more highly processed, complex- types in glycopeptides containing the H48 allotype as compared to L48. A) The top 10 N45 glycopeptides identified on the L48 allele specific glycopeptide from all monocyte donors includes 6 hybrid, 2 complex and 2 oligomannose type N-glycans based on relative intensity. Percent composition of each type breaks down to 68% hybrid, 23% complex and 9% oligomannose type. B) The top 10 N45 glycopeptides identified on the H48 allele specific glycopeptide include 5 hybrid and 5 complex-type N-glycans based on relative intensity. Percent composition of each type breaks down to 62% hybrid and 38% complex-type. A-B) The glycopeptide red N is N45 while the L/H48 residue is labeled cyan in the N45 glycopeptide identified. Relative abundance is based on relative intensities.
Discussion
CD16a H48 displays more processed, complex-type N-glycans than CD16a L48 from monocytes at site N45. Hybrid and oligomannose-type N-glycans total 77% and
23% for complex-type on the L48 specific glycopeptide at N45 while the H48 specific glycopeptide at N45 displays 62% hybrid and 38% complex-type. Furthermore, two tetraantennerary complex-types are in the top 10 of the H48 specific glycopeptide indicating highly processed forms not seen for L48. Interestingly, CD16a isolated from an NK cell donor heterozygous for H/L48 also displayed similar increased processing at 132 the CD16a H48 specific N45 glycopeptide as compared to L48 (data not shown, 12).
Additionally, this difference in N-glycosylation was only observed on the N45 glycosite.
This is likely because the L/H48 residue resides near the N45 loop thus it is likely this residue can affect loop dynamics affecting the N45 N-glycan mobility. Increased mobility of the N-glycan would likely make the N-glycan more accessible to glycosyl transferases allowing more processing of the N-glycan. However, more studies on the role of L/H48 concerning N-glycan mobility and processing need to be performed to fully understand the effect of H48. The shift in CD16a N-glycosylation processing at N45 from monocytes and NK cells may be a modest one, however this data adds to the molecular characterization of the disease states in homozygous individuals for the H48 allele (4-5).
Works Cited
1. Yeap, W. H. et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci. Rep. 6, 34310 (2016). 2. Subedi, G. P. & Barb, A. W. CD16a with oligomannose-type N-glycans is the only ‘low-affinity’ Fc receptor that binds the IgG crystallizable fragment with high affinity in vitro. J. Biol. Chem. 293, 16842–16850 (2018). 3. Patel, K. R., Roberts, J. T., Subedi, G. P. & Barb, A. W. Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function. J. Biol. Chem. jbc.RA117.001207 (2018). doi:10.1074/jbc.RA117.001207 4. Jawahar, S. et al. Natural Killer (NK) cell deficiency associated with an epitope- deficient Fc receptor type IIIA (CD16-II). Clin. Exp. Immunol. 103, 408–413 (1996). 5. de Vries, E. et al. Identification of an unusual Fc gamma receptor IIIa (CD16) on natural killer cells in a patient with recurrent infections. Blood 88, 3022–3027 (1996). 6. Mace, E. M. & Orange, J. S. Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets. Front. Immunol. 7, 545 (2016). 7. de Haas, M. et al. A triallelic Fc gamma receptor type IIIA polymorphism influences the binding of human IgG by NK cell Fc gamma RIIIa. J. Immunol. Baltim. Md 1950 156, 2948–2955 (1996). 133
8. Mahaweni, N. M. et al. A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism. Sci. Rep. 8, 1–11 (2018). 9. Musolino, A., Naldi, N., Bortesi, B., Pezzuolo, D., Capelletti, M., Missale, G., Laccabue, D., Zerbini, A., Camisa, R., Bisagni, G., Neri, T. M., and Ardizzoni, A. (2008) Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J Clin Oncol 26, 1789-1796 10. Weng, W. K., and Levy, R. (2003) Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol 21, 3940-3947 11. Grier, J. T. et al. Human immunodeficiency-causing mutation defines CD16 in spontaneous NK cell cytotoxicity. J. Clin. Invest. 122, 3769–3780 (2012). 12. Patel, K. R., Nott, J. D. & Barb, A. W. Primary human natural killer cells retain proinflammatory IgG1 at the cell surface and express CD16a glycoforms with donor- dependent variability. Mol. Cell Proteomics (2019). doi:10.1074/mcp.RA119.001607 134
APPENDIX B. INTRADOMAIN INTERACTIONS IN AN NMDA RECEPTOR FRAGMENT MEDIATE N-GLYCAN PROCESSING AND CONFORMATIONAL SAMPLING
A manuscript in Structure. Doi: 10.1016/j.str.2018.09.010
The following sections are the contributions by JTR to the manuscript cited above.
Introduction
The N-methyl-D-aspartate receptor (NMDAR) is an important ionotropic glutamate receptor which is a ligand-gated ion channel and essential to synaptic transmission in the central nervous system (1-3). Neurotransmitter receptors like
NMDAR have been described to be important in learning, memory, development and synaptic plasticity (4-6). NMDARs are integral membrane heterotetramers made up of 2
GluN1 subunits and 2 GluN2 (A-D) or GluN3(A/B) subunits (7-9). Each of these subunits contain a ligand binding domain (LBD), transmembrane domain, intracellular
C-terminal domain and amino-terminal domain (ATD). The GluN1 subunit is heavily glycosylated with up to 11 N-glycosylation sites in the extracellular domain (10-12). N- glycosylation sequons are highly conserved amongst NMDARs and are expected to be functionally important. Indeed, N-glycosylation is essential for proper oligomerization
(14, 15) and has been described to stabilize glycine binding (16, 17)). NMDAR N- glycosylation of recombinantly expressed material has not been identified. This study identifies the N-glycosylation profile of the GluN1-LBD domain expressed in HEK293f cells. Other studies that are part of the published manuscript not discussed here use an 135 approach encompassing NMR, MD and MS methods to correlate N-glycosylation site- specific processing of GluN1-LBD to intramolecular carbohydrate/polypeptide contacts and protein structure.
Experimental Procedures
Procainamide labeling of PNGase F-released N-glycans
Following heat denaturation of 50 μg recombinant GluN1 LBD for 5 min at 90 °C,
1 µL sequencing grade trypsin (Promega; 1 mg/mL in 50 mM ammonium carbonate, pH
8.0) was added and incubated at 37 °C for 18 h after mixing. The trypsinized sample was again incubated at 90 °C for 5 min to denature the trypsin, followed by cooling for 5 min on ice. PNGase F (1 µL; glycerol-free; New England BioLabs) was then added to the sample and incubated at 37 °C for 18 h. A Hypersep C18 column (ThermoFisher) was washed with 3 × 1 mL methanol then 3 × 1 mL 5% acetic acid. Free N-glycans were isolated by applying the sample, diluted in 5% acetic acid, over the C18 column and collecting the flow through. Wash fractions (3 × 1 mL 5% acetic acid were collected in the same tube, then frozen and lyophilized. The lyophilized sample was resuspended in 200 µL ddH2O by vortexing and then transferred to a microcentrifuge tube, frozen, and lyophilized. The sample was then resuspended in 10 µL of ddH2O. A stock solution containing 2 M sodium cyanoborohydride, 1 M procainamide and 50% acetic acid (10
µL) was added to the sample, mixed and incubated for 18 h at 37 °C. The sample was then lyophilized and resuspended into 10 µL ddH2O .
136
Hydrophilic interacting chromatography mass spectrometry
The HPLC instrument used was an Agilent 1260 HPLC with an Acquity UPLC
Glycoprotein BEH Amide, 300 Å, 1.7 µm Hydrophilic Interaction Liquid Chromatography
(HILIC) column (Waters). The lyophilized procainamide-labeled glycan samples were re-suspended in ddH2O and were injected (3 µL; corresponding to 15 μg of GluN1 LBD) onto the column prewashed with 25% Solvent A (0.1% formic acid and 0.01% trifluoracetic acid in ddH2O) and 75% Solvent B (0.1% formic acid and 0.01% trifluoracetic acid in acetonitrile) at 0.1 mL/min. The glycans were then eluted by a gradient at 0.1 mL/min flow rate unless indicated otherwise; 0.5-10.5 minutes (gradient from 25%-35% A), 10.5-40.5 minutes (a gradient from to 35% to 50% A), 40.5-41.5 minutes (gradient to 100% A), 41.5-43.5 minutes (100% A), 46.5-47 minutes (25% A,
75% B) at 0.05 mL/min and 47-60 minutes (25% A, 75% B) at 0.1 mL/min at 45 °C.
Upon elution, the samples were coupled in-line to a Q Exactive Hybrid Quadrupole-
Orbitrap Mass Spectrometer (Thermo Scientific). The mass spectrometer (scan range
266.7- 4000 m/z) subjected ions to HCD fragmentation (scan range of 200-2000 m/z) at a normalized collisional energy of 27 eV. In between samples, identical chromatography and MS analyses were performed following an injection of water to ensure no carryover between samples. Samples spectra were then analyzed by running the software Byonic
(Protein Metrics) from the RAW spectra file. These matches were then validated manually by analyzing the RAW file in XCaliber (Thermo Scientific). Average mass error for each data set was calculated according to Equation 1.
137
∑푛|푚/푧푐푎푙푐− 푚/푧표푏푠| 푒푟푟표푟 = 1 푛 푛 Eq. 1 푛
Results
Glycomics of the recombinant GluN1 LBD
Glycan remodeling may provide additional insight into N-glycan/polypeptide interactions. The predominant GluN1 glycoforms are expected to be of an oligomannose-type (13,14) however, it is unclear if the factors that restrict N-glycan processing in neuronal cells will be present when expressing the isolated GluN1 LBD with HEK 293F cells. It is noteworthy that the N-glycan NMR peaks observed using the
GluN1 LBD expressed from HEK293F cells (with the capacity to generate highly- processed complex-type N-glycans) were comparable to the same peaks from the
GluN1 LBD expressed from HEK293S (lec1-/-) cells (that expresses protein with primarily Man5 N-glycans;). This comparability indicates that N-glycan composition minimally impacted the chemical environment for these 1H-13C correlations. To study the global N-glycan composition in the recombinant GluN1 LBD expressed with the
HEK293F cell line, N-glycans were released by PNGase F treatment and derivatized using procainamide for mass spectrometric analyses. We identified 52 unique N-glycan species that classified into 1.9% hybrid, 13.5% oligomannose and 84.6% complex types with an average mass error between the calculated mass and observed mass was
0.007 Da (Figure B.1A). Thus, the N-glycans found on the GluN1 LBD expressed in
HEK293F cells are different from those expected on neuronal NMDARs. This presents an alternative opportunity to probe intramolecular N-glycan interactions by assessing the extent of N-glycan remodeling. For example, the complete absence of terminal sialic 138 acid modifications potentially indicates interactions between non-reducing terminal carbohydrate residues that prevent sialic acid addition in a cell line that is capable of heavily sialylating N-glycans (19,20).
The subset of complex-type glycans were further characterized to reveal biantennary (32%), triantennary (32%) and tetraantennary or greater (36%) forms
(Figure B.1A). Fucose was found attached to 65.9% of complex, 100% of hybrid and
14.3% of oligomannose types (Figure B.1B). N-acetylneuraminic acid (a sialic acid) modified 70.5% of complex type, 0% of oligomannose and 0% of hybrid glycoforms.
These data identify a distribution of N-glycan composition and the presence of a majority of highly processed glycoforms. Therefore, the N-glycans in the recombinant
GluN1 fragment are exposed to glycan modifying enzymes in the ER and Golgi and factors restricted to the expressed fragment do not limit N-glycan processing to the oligomannose forms identified on the GluN1 subunit from native tissue (13).
Figure B.1. Procainamide-derivatized free N-glycan composition of recombinant GluN1-NMDA expressed from HEK293F cells (JTR). A) Relative abundance of the N-glycans identified are characterized as hybrid, oligomannose and complex. Complex-type is further split into bi, tri and tetra+ antennary complex N- glycans. B) % fucosylated N-glycans identified within each N-glycan type. 139
Discussion
Mass Spectrometry of N-glycans, molecular dynamics (MD) simulations and
NMR correlated N-glycan composition with polypeptide-carbohydrate interactions. A recently developed NMR 2d one-bond proton (1H) and carbon (13C) correlation experiment was used to identify these contacts. Here we defined the N-glycan composition of recombinant GluN1-NMDAR using glycomics. Furthermore, glycoproteomics showed differential processing depending on N-glycosylation site.
Interestingly, MD simulations observed intramolecular contacts of the carbohydrates and polypeptide which help explain differences in GluN1-NMDAR site-specific N- glycosylation processing. These intramolecular contacts within GluN1-NMDAR were hypothesized to limit processing by glycan modifying enzymes.
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