INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

Clinical and Translational Glycoscience Research Center Georgetown University Medical Center Georgetown University Washington DC USA

Department of Biochemistry and Molecular and Cellular Biology Georgetown University Washington DC USA

Department of Oncology Lombardi Comprehensive Cancer Center Georgetown University Washington DC USA

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Vestec Czech Republic

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Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–30. PubMed

Liu JL, Patel HD, Haney NM, Epstein JI, Partin AW. Advances in the selection of patients with prostate cancer for active surveillance. Nat Rev Urol. 2021;18(4):197–208. PubMed

Haffner MC, Zwart W, Roudier MP, et al. Genomic and phenotypic heterogeneity in prostate cancer. Nat Rev Urol. 2021;18(2):79–92. PubMed PMC

Oesterling JE, Chan DW, Epstein JI, et al. Prostate specific antigen in the preoperative and postoperative evaluation of localized prostatic cancer treated with radical prostatectomy. J Urol. 1988;139(4): 766–772. PubMed

Catalona WJ, Partin AW, Slawin KM, et al. Use of the percentage of free prostate-specific antigen to enhance differentiation of prostate cancer from benign prostatic disease: a prospective multicenter clinical trial. JAMA. 1998;279(19):1542–1547. PubMed

Sartori DA, Chan DW. Biomarkers in prostate cancer: what’s new? Curr Opin Oncol. 2014;26(3):259–264. PubMed PMC

Haga Y, Uemura M, Baba S, et al. Identification of multisialylated LacdiNAc structures as highly prostate cancer specific glycan signatures on PSA. Anal Chem. 2019;91(3):2247–2254. PubMed

Llop E, Ferrer-Batallé M, Barrabés S, et al. Improvement of prostate cancer diagnosis by detecting PSA glycosylation-specific changes. Theranostics. 2016;6(8):1190–1204. PubMed PMC

Munkley J, Mills IG, Elliott DJ. The role of glycans in the development and progression of prostate cancer. Nat Rev Urol. 2016;13(6): 324–333. PubMed

Tkac J, Gajdosova V, Hroncekova S, et al. Prostate-specific antigen glycoprofiling as diagnostic and prognostic biomarker of prostate cancer. Interface Focus. 2019;9(2):20180077. PubMed PMC

Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science. 2001;291(5512):2364–2369. PubMed

Schjoldager KT, Narimatsu Y, Joshi HJ, Clausen H. Global view of human protein glycosylation pathways and functions. Nat Rev Mol Cell Biol. 2020;21(12):729–749. PubMed

Cummings RD. The repertoire of glycan determinants in the human glycome. Mol BioSyst. 2009;5(10):1087–1104. PubMed

Tran DT, Ten Hagen KG. Mucin-type O-glycosylation during development. J Biol Chem. 2013;288(10):6921–6929. PubMed PMC

Varki A Biological roles of glycans. Glycobiology. 2017;27(1):3–49. PubMed PMC

Dennis JW, Nabi IR, Demetriou M. Metabolism, cell surface organization, and disease. Cell. 2009;139(7):1229–1241. PubMed PMC

Macauley MS, Crocker PR, Paulson JC. Siglec-mediated regulation of immune cell function in disease. Nat Rev Immunol. 2014;14(10): 653–666. PubMed PMC

RodrIguez E, Schetters STT, van Kooyk Y. The tumour glyco-code as a novel immune checkpoint for immunotherapy. Nat Rev Immunol. 2018;18(3):204–211. PubMed

Kawahara R, Recuero S, Srougi M, et al. The complexity and dynamics of the tissue glycoproteome associated with prostate cancer progression. Mol Cell Proteomics. Published online January 05, 2020. PubMed PMC

Petrosyan A, Holzapfel MS, Muirhead DE, Cheng PW. Restoration of compact Golgi morphology in advanced prostate cancer enhances susceptibility to galectin-1-induced apoptosis by modifying mucin O-glycan synthesis. Mol Cancer Res. 2014;12(12):1704–1716. PubMed PMC

Tzeng SF, Tsai CH, Chao TK, et al. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer. FASEB J. 2018:fj201800687. PubMed

Murphy K, Murphy BT, Boyce S, et al. Integrating biomarkers across omic platforms: an approach to improve stratification of patients with indolent and aggressive prostate cancer. Mol Oncol. 2018;12(9): 1513–1525. PubMed PMC

Wang X, Chen J, Li QK, et al. Overexpression of alpha (1,6) fucosyltransferase associated with aggressive prostate cancer. Glycobiology. 2014;24(10):935–944. PubMed PMC

Bařinka C, Rojas C, Slusher B, Pomper M. Glutamate carboxypeptidase II in diagnosis and treatment of neurologic disorders and prostate cancer. Curr Med Chem. 2012;19(6):856–870. PubMed PMC

Barinka C, Sácha P, Sklenár J, et al. Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity. Protein Sci. 2004;13(6):1627–1635. PubMed PMC

Holmes EH, Greene TG, Tino WT, et al. Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients. Prostate Suppl. 1996;7:25–29. PubMed

Rovenská M, Hlouchová K, Sácha P, et al. Tissue expression and enzymologic characterization of human prostate specific membrane antigen and its rat and pig orthologs. Prostate. 2008;68(2): 171–182. PubMed

Sácha P, Zámecník J, Barinka C, et al. Expression of glutamate carboxypeptidase II in human brain. Neuroscience. 2007;144(4):1361–1372. PubMed

Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3(1):81–85. PubMed

Vornov JJ, Peters D, Nedelcovych M, Hollinger K, Rais R, Slusher BS. Looking for drugs in all the wrong places: use of GCPII inhibitors outside the brain. Neurochem Res. 2020;45(6):1256–1267. PubMed PMC

Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: a study of 184 cases. Cancer. 1998; 82(11):2256–2261. PubMed

Afshar-Oromieh A, Babich JW, Kratochwil C, et al. The rise of PSMA ligands for diagnosis and therapy of prostate cancer. J Nucl Med. 2016;57(Suppl 3):79S–89S. PubMed

Cho SY, Gage KL, Mease RC, et al. Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer. J Nucl Med. 2012;53(12):1883–1891. PubMed PMC

Evans MJ, Smith-Jones PM, Wongvipat J, et al. Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen. Proc Natl Acad Sci U S A. 2011;108(23):9578–9582. PubMed PMC

Ristau BT, O’Keefe DS, Bacich DJ. The prostate-specific membrane antigen: lessons and current clinical implications from 20 years of research. Urol Oncol. 2014;32(3):272–279. PubMed PMC

Zaorsky NG, Yamoah K, Thakur ML, et al. A paradigm shift from anatomic to functional and molecular imaging in the detection of recurrent prostate cancer. Future Oncol. 2014;10(3):457–474. PubMed PMC

Werner RA, Derlin T, Lapa C, et al. F-18-labeled, PSMA-targeted radiotracers: leveraging the advantages of radiofluorination for prostate cancer molecular imaging. Theranostics. 2020;10(1):1–16. PubMed PMC

Kopka K, Benešová M, Bařinka C, Haberkorn U, Babich J. Glu-Ureido-based inhibitors of prostate-specific membrane antigen: lessons learned during the development of a novel class of low-molecular-weight theranostic radiotracers. J Nucl Med. 2017;58: 17s–26s. PubMed

Lütje S, Heskamp S, Cornelissen AS, et al. PSMA ligands for radionuclide imaging and therapy of prostate cancer: clinical status. Theranostics. 2015;5(12):1388–1401. PubMed PMC

Fendler WP, Calais J, Eiber M, et al. Assessment of Ga-68-PSMA-11 PET accuracy in localizing recurrent prostate cancer: a prospective single-arm clinical trial. Jama Oncology. 2019;5(6):856–863. PubMed PMC

Rowe SP, Gorin MA, Pomper MG. Imaging of prostate-specific membrane antigen with small-molecule PET radiotracers: from the bench to advanced clinical applications. Annu Rev Med. 2019;70(70): 461–477. PubMed

Hofman MS, Lawrentschuk N, Francis RJ, et al. Prostate-specific membrane antigen PET-CT in patients with high-risk prostate cancer before curative-intent surgery or radiotherapy (proPSMA): a prospective, randomised, multicentre study. Lancet. 2020;395(10231):1208–1216. PubMed

Perera M, Papa N, Roberts M, et al. Gallium-68 prostate-specific membrane antigen positron emission tomography in advanced prostate cancer-updated diagnostic utility, sensitivity, specificity, and distribution of prostate-specific membrane antigen-avid lesions: a systematic review and meta-analysis. Eur Urol. 2020;77(4):403–417. PubMed

Turpin A, Girard E, Baillet C, et al. Imaging for metastasis in prostate cancer: a review of the literature. Front Oncol. 2020;10:55. PubMed PMC

Hofman MS, Emmett L, Sandhu S, et al. [Lu-177]Lu-PSMA-617 versus cabazitaxel in patients with metastatic castration-resistant prostate cancer (TheraP): a randomised, open-label, phase 2 trial. Lancet. 2021;397(10276):797–804. PubMed

Kratochwil C, Bruchertseifer F, Giesel FL, et al. 225Ac-PSMA-617 for PSMA-targeted alpha-radiation therapy of metastatic castration-resistant prostate cancer. J Nucl Med. 2016;57(12):1941–1944. PubMed

Kratochwil C, Giesel FL, Stefanova M, et al. PSMA-targeted radionuclide therapy of metastatic castration-resistant prostate cancer with 177Lu-labeled PSMA-617. J Nucl Med. 2016;57(8): 1170–1176. PubMed

Kratochwil C, Haberkorn U, Giesel FL. Radionuclide therapy of metastatic prostate cancer. Semin Nucl Med. 2019;49(4):313–325. PubMed

Liu T, Toriyabe Y, Berkman CE. Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography. Protein Expr Purif. 2006;49(2):251–255. PubMed

Barinka C, Ptacek J, Richter A, Novakova Z, Morath V, Skerra A. Selection and characterization of Anticalins targeting human prostate-specific membrane antigen (PSMA). Protein Eng Des Sel. 2016;29(3):105–115. PubMed

Skultetyova L, Ustinova K, Kutil Z, et al. Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules. Sci Rep. 2017;7(1):11547. PubMed PMC

Nováková Z, Foss CA, Copeland BT, et al. Novel monoclonal antibodies recognizing human prostate-specific membrane antigen (PSMA) as research and theranostic tools. Prostate. 2017;77(7): 749–764. PubMed PMC

Yang W, Shah P, Hu Y, et al. Comparison of enrichment methods for intact N- and O-linked glycopeptides using strong anion exchange and hydrophilic interaction liquid chromatography. Anal Chem. 2017; 89(21):11193–11197. PubMed PMC

Benicky J, Sanda M, Pompach P, Wu J, Goldman R. Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease. Anal Chem. 2014;86(21):10716–10723. PubMed PMC

Yuan W, Benicky J, Wei R, Goldman R, Sanda M. Quantitative analysis of sex-hormone-binding globulin glycosylation in liver diseases by liquid chromatography-mass spectrometry parallel reaction monitoring. J Proteome Res. 2018;17(8):2755–2766. PubMed PMC

Horoszewicz JS, Leong SS, Kawinski E, et al. LNCaP model of human prostatic carcinoma. Cancer Res. 1983;43(4):1809–1818. PubMed

Sanda M, Benicky J, Goldman R. Low collision energy fragmentation in structure-specific glycoproteomics analysis. Anal Chem. 2020; 92(12):8262–8267. PubMed PMC

Sanda M, Goldman R. Data independent analysis of IgG glycoforms in samples of unfractionated human plasma. Anal Chem. 2016; 88(20):10118–10125. PubMed PMC

Depraz Depland A, Renois-Predelus G, Schindler B, Compagnon I. Identification of sialic acid linkage isomers in glycans using coupled InfraRed Multiple Photon Dissociation (IRMPD) spectroscopy and mass spectrometry. Int J Mass Spectrom. 2018;434:65–69.

Pavlicek J, Ptacek J, Barinka C. Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme. Curr Med Chem. 2012;19(9):1300–1309. PubMed

Wright GL Jr., Haley C, Beckett ML, Schellhammer PF. Expression of prostate-specific membrane antigen in normal, benign, and malignant prostate tissues. Urol Oncol. 1995;1(1):18–28. PubMed

Barinka C, Rinnová M, Sácha P, et al. Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J Neurochem. 2002;80(3):477–487. PubMed

Christiansen JJ, Rajasekaran SA, Inge L, et al. N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy. Mol Cancer Ther. 2005;4(5):704–714. PubMed

Ghosh A, Heston WD. Effect of carbohydrate moieties on the folate hydrolysis activity of the prostate specific membrane antigen. Prostate. 2003;57(2):140–151. PubMed

Mesters JR, Barinka C, Li W, et al. Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. EMBO J. 2006;25(6):1375–1384. PubMed PMC

Ghafoor S, Burger IA, Vargas AH. Multimodality imaging of prostate cancer. J Nucl Med. 2019;60(10):1350–1358. PubMed PMC

Han S, Woo S, Kim YJ, Suh CH. Impact of Ga-68-PSMA PET on the management of patients with prostate cancer: a systematic review and meta-analysis. Eur Urol. 2018;74(2):179–190. PubMed

Berger UV, Carter RE, McKee M, Coyle JT. N-acetylated alpha-linked acidic dipeptidase is expressed by non-myelinating Schwann cells in the peripheral nervous system. J Neurocytol. 1995;24(2):99–109. PubMed

Knedlík T, Navrátil V, Vik V, Pacík D, Šácha P, Konvalinka J. Detection and quantitation of glutamate carboxypeptidase II in human blood. Prostate. 2014;74(7):768–780. PubMed

Schülke N, Varlamova OA, Donovan GP, et al. The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy. Proc Natl Acad Sci U S A. 2003;100(22):12590–12595. PubMed PMC