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Antibodies Encoded Natural Igm − Ligands for VH4-34 Poly-N-Acetyl

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Antibodies Encoded Natural Igm − Ligands for VH4-34 Poly-N-Acetyl Identification of Cell Surface Straight Chain Poly-N-Acetyl-Lactosamine Bearing Protein Ligands for VH4-34−Encoded Natural IgM Antibodies This information is current as of September 24, 2021. Neelima M. Bhat, Christopher M. Adams, Yi Chen, Marcia M. Bieber and Nelson N. H. Teng J Immunol 2015; 195:5178-5188; Prepublished online 26 October 2015; doi: 10.4049/jimmunol.1501697 Downloaded from http://www.jimmunol.org/content/195/11/5178 Supplementary http://www.jimmunol.org/content/suppl/2015/10/24/jimmunol.150169 http://www.jimmunol.org/ Material 7.DCSupplemental References This article cites 80 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/195/11/5178.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Identification of Cell Surface Straight Chain Poly-N-Acetyl- Lactosamine Bearing Protein Ligands for VH4-34–Encoded Natural IgM Antibodies Neelima M. Bhat,* Christopher M. Adams,† Yi Chen,* Marcia M. Bieber,* and Nelson N. H. Teng* B cell binding and cytotoxicity by human VH4-34–encoded Abs of the IgM isotype has been well documented. A VH4-34-IgM has recently shown a favorable early response in a phase 1 trial for treatment of B cell acute lymphoblastic leukemia. Although its B cell ligand has been identified as straight chain poly-N-acetyl-lactosamine (SC-PNAL), the carrier of the sugar moiety has not been identified. Using nanoelectrospray ionization mass spectrometry, we identify the metabolic activation related protein com- plex of CD147-CD98 as a major carrier of poly-N-acetyl-lactosamine (SC-PNAL) on human pre-B cell line Nalm-6. Previous Downloaded from studies have suggested CD45 as the SC-PNAL carrier for VH4-34–encoded IgG Abs. Because Nalm-6 is CD45 negative, human peripheral blood B lymphocytes and human B cell line, Reh, with high CD45 expression, were examined for SC-PNAL carrier proteins. Western blot analysis shows that the CD147-98 complex is indeed immunoprecipitated by VH4-34–encoded IgMs from human peripheral blood B lymphocytes and human B cell lines, Reh, OCI-Ly8, and Nalm-6. However, CD45 is immunoprecipi- tated only from peripheral B lymphocytes, but not from Reh despite the high expression of CD45. These results suggest that human B cells retain SC-PNAL on the CD147-98 complex, but modulate the sugar moiety on CD45. Because the carbohydrate http://www.jimmunol.org/ moiety may act as a selecting Ag for VH4-34 autoantibody repertoire, its differential expression on proteins may provide a clue to the intricate atypical regulation of the VH4-34 gene. The Journal of Immunology, 2015, 195: 5178–5188. ntibodies encoded by the VH4-34 gene have been studied nasopharyngeal carcinoma and a subset of cold agglutinin disease because of their inherent autoreactivity and unusual (5–11). The shared feature among these diverse syndromes is as- A regulation. B lymphocytes bearing the VH4-34-BCR are sociation with lymphotropic viruses and B cell hyperproliferation (6). overrepresented in the naive repertoire but counterselected for Ab In IM, a widespread disease caused by EBV, the secreted VH4-34 secretion. Although nearly 5% of naive B cells use the V4-34-BCR response is transitory and limited to the multivalent IgM and IgA by guest on September 24, 2021 circulating Abs are low to undetectable in healthy adults (1, 2). In disappearing with the resolution of the disease (12). No manifestation fact, VH4-34-BCR bearing cells exhibit a surface profile of anergic of long-term clinical autoimmunity is linked with brief secretion of cells, are actively policed away from germinal centers in healthy high-avidity VH4-34 Abs approaching 400 mg/ml. Isotype-switched adults, and are primarily found in the marginal or mantle zone (3, 4). VH4-34 Abs are detected in .50% of SLE patients (13). The op- Circulating VH4-34 Abs are readily detectable in limited clinical erative mechanism that restricts VH4-34-BCR bearing B cells from conditions, such as infectious mononucleosis (IM), AIDS, systemic entering germinal center reactions is blocked, leading to the pro- lupus erythematosus (SLE), hepatitis C infection, EBV-associated duction of isotype-switched somatically mutated autoantibodies (3). Hence, secretion of VH4-34 Abs has been monitored to study the *Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, interplay between acute anti-pathogen response and breakdown in Stanford University, Stanford, CA 94305; and †Stanford University Mass Spectrom- tolerance leading to autoimmunity, particularly in SLE. etry, Stanford University, Stanford, CA 94305 In the context of autoreactivity, it is the recognition of a con- ORCIDs: 0000-0002-6472-1638 (N.M.B.); 0000-0002-0210-3711 (C.M.A.). served carbohydrate epitope on human adult or fetal RBC and Received for publication July 29, 2015. Accepted for publication September 24, B lymphocytes that sets VH4-34 Abs apart from the typical natural 2015. autoantibodies. The germ-line nonmutated framework region of This work was supported by the Malloy Research Gift Fund, Division of Gynecologic the VH4-34 H chain, independent of the L chain, is accountable Oncology, Department of Obstetrics and Gynecology, Stanford University; the re- sources and collaborative efforts provided by the Consortium for Functional Glyco- for binding linear/straight chain poly-N-acetyl-lactosamine (SC- mics were funded by NIGMS-GM62116. PNAL) or i-antigen on human fetal/nucleated RBC and human Address correspondence and reprint requests to Dr. Neelima M. Bhat, HH333, Di- B lymphocytes. Despite the preponderance of circulating i-antigen/ vision of Gynecologic Oncology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5317. E-mail SC-PNAL on fetal RBCs, B lymphocytes bearing VH4-34 BCR address: [email protected] are positively selected in human fetal immune repertoire. This scenario The online version of this article contains supplemental material. represents an unprecedented selection of an autoreactive BCR in the Abbreviations used in this article: CFG, Consortium for Functional Glycomics; DIF, presence of abundant autoantigen, making the VH4-34 gene a rare detergent insoluble fraction; D-threo PDMP, 1-phenyl-2-decanoylamino-3-morpholino- example where expression of surface versus secreted Ig is stringently 1-propanol, HCl; FB1, fumonisin B1; FDR, false discovery rate; GO, gene ontology; HG-CD147, high glycoform CD147; IM, infectious mononucleosis; IP, immunoprecip- regulated. In contrast, the branched SC-PNAL chains or i-antigen is itation; LC, liquid chromatography; LG-CD147, low glycoform-CD147; Nano-ESI-MS, present only on adult human RBCs and are not bound by anti–B cell nanoelectrospray ionization mass spectrometry; RT, room temperature; SC-PNAL, germ line–encoded VH4-34 IgM Abs (14). poly-N-acetyl-lactosamine; SLE, systemic lupus erythematosus; TL, tomato lectin. The anti-i/anti–B cell SC-PNAL binding VH4-34 Abs, range Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 from high to low binders, with the high binders demonstrating www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501697 The Journal of Immunology 5179 B cell cytotoxicity via membrane perturbations. Such VH4-34 Abs and results can be accessed at www.functionalglycomics.org under CFG are observed to bind or paint B lymphocytes in patients with IM, request number 2112. SLE, or HIV (15, 16). On RBCs, SC-PNAL is present on both TLC glycolipids and glycoproteins (17). The nature of the biological backbone that carries the sugar epitope on B lymphocytes has not Glycans on a ceramide backbone were purified as described (20, 21), separated on TLC, and immunostained as described (22, 23). Plate was air been studied in detail. Earlier work with VH4-34-IgG has iden- dried and blocked with PBS with 5% BSA for 2 h and exposed overnight tified CD45 protein as a carrier of SC-PNAL (18). In this study, with mAb 216 (5 mg/ml). After washing five times with 0.5% BSA/PBS, we show that SC-PNAL is present on CD147 and other proteins the plate was incubated for l h with 125I-labeled anti-human IgM (Zymed known to associate with it, such as CD98. This membrane super Laboratories, South San Francisco, CA). After 10 washes with PBS, the plate was then air-dried and subjected to autoradiography. Total staining of complex plays a central role in cell proliferation, transport, and glycan-ceramides was performed by orcinol-H2SO4 as described (20, 21). energy metabolism. SC-PNAL expression on CD45 is detected Glycan-ceramide purification and TLC studies were performed by Dr. Sen-itiroh only on peripheral blood B lymphocytes and not on human B cell Hakomori (University of Washington, Seattle, WA). lines, suggesting differential glycosylation patterns. Because gly- Abs and reagents can moieties have crucial functions from protein folding/adhesion to signaling, differential expression of SC-PNAL on proteins in VH4-34 gene–encoded MAb 216, Z21, Z2D2, and A6H4C5 have been different B cell types may be linked to the intricate control of described previously (14).
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