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O-glycosylation and protein evolution: the case of the LHb to CGb development David Ben-Menahem Clinical Biochemistry and Pharmacology, Faculty of Health Sciences Ben-Gurion University of the Negev, Beer-Sheva, Israel Structure-Function of the Gonadotropins; members of the glycoprotein hormone family • Lutropin (LH), follitropin (FSH) are expressed in the pituitary and Choriogonadotropin (CG) is synthesized in the placenta of primates and equids • Non-covalent heterodimers composed of a common α subunit and a hormone-specific β subunit. Only dimers are active; monomeric subunits do not bind to the cognate receptor. Both LH and CG activate the LH/CG receptor (LHR) The Gonadotropin Subunits α NH2 1 FSHb NH2 1 LHb NH2 1 52 7 24 30 Primates/Equids hCGb NH2 78 92 COOH 110 COOH 121 COOH o o o o 1 13 30 COOH 145 CTP The LHb to CGb subunit development; Carboxy Terminal Peptide extention (CTP) characteristics FS hLHb NH2 1 30 COOH 121 o o o o hCGb NH2 1 13 145 COOH 30 CTP • The CGb gene presumably evolved from the ancestral LHb gene • Ser/Thr/Pro rich domain, multiple O-glycans attached to the CTP (4-12) • Prolongs circulatory survival compared to LH • Orient secretion of hCG from the apical side of placental trophoblasts into the maternal circulation to delay luteolysis in primates Why the CTP domain is not wide-spread in the animal kingdom? This is intriguing because the LHb gene is conserved among mammals, few mutations localized to a small region and the gain of new hormonal properties Whether the LHb genes in species other than primates and equids contain an untranslated CTP-like sequence? Yes, a CTP-like sequence is cryptic in the LHb gene of several mammals but not in birds, amphibians and fishes Whether the incorporation of the cryptic CTP sequence in the bovine LHb reading frame will result in misfolding and degradation or allow the expression of the extended subunit? bLHβ: 110 CDHPPLPDILFL121 bLHβboCTP: 110CD....P…QTSSSSKDAPLQP...PMPILTLQTSRHSS PPFPIKTS147 eLH/CGβ: 110CA....P…QASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTS149 hCGβ: 110CDDPRFQASSSSKAPPP...SLPSPSRL...PGPSDTPILPQ145 Nakav et al., 2005 Expression and secretion of the bovine elongated LHβboCTP subunit in transfected CHO cells FS 1 111 1 121 LHβ 1 + D 121 1 LHβ Nakav et al., 2005 LHβ boCTP 121 LHβ 147 LHβ 111 1 LHβ 142 111 1 huCTP LHβboCTP LHβ111 huCTP LHβCTP Structure and Function of the boCTP Domain 110CDHPQLSGLLFL121 hLHb: hCGb (bwt): 110CDDPRFQASSSSKAPPPSLPSPSRLPGPSDTPILPQ145 hCGbboCTP (bboCTP) 110CDDPRFQASSSSKDAPLQPPMPILTLQTSRHSSPPFPIKTS150 hCGb117 (b117) 110CDDPRFQA117 CGb Cloned in PM2 and stably transfected into CHO cells Secretion kinetics: Pulse Chase analysis 1 117 hCGb b wt Recovery (%): t½ (min): Nakav et al., 2005 65 ± 5 80 ± 5 145 CTP 1 hCGb 150 boCTP bboCTP 65 ± 5 115 ± 10 1 117 hCGb b117 50 ± 5 90 ± 5 Gabay et al., 2014 Sialic Acid (4) Sialic Acid (1) Gal\GalNAc (8) Gal\GalNAc (7) Gal\GalNAc (6) Gal\GalNAc (5) CGβboCTP Gal\GalNAc (4) Gal\GalNAc (3) Gal\GalNAc (2) o Gal\GalNAc (1) oo o Beta Gal (2) CTP Beta Gal (1) Alpha Gal (1) Terminal GlcNAc (1) CGβWT Fucose (6) Mannose (3) Mannose (2) Glc/Man (3) Glc/Man (2) 30000 Glc/Man (1) 35000 GlcNAc (1) 40000 Complex (4) 45000 Complex (3) Complex (1) Fluorescence (A.U.) Lectin array analysis of the secreted chimeric subunit; absence of mucin type O-glycans CGβ117 ? boCTP CGβ CGβboCTP CGβ117 25000 20000 15000 10000 5000 0 Basolateral secretion of the CGbboCTP chimera from polarized MDCK cells Apical (%) Basolateral (%) CGbboCTP 30 hCGb 70 boCTP Boime and his colleagues Apical (%) Basolateral (%) CGb o o o o 1 13 30 CTP 65 35 25 75 145 LHb 1 30 121 CGb - Odg 20 1 13 30 80 145 CTP Nakav et al., 2005 ) Plasma Concentration subunit (ng/ml)(ng/ml) Pharmacokinetics of the CGβboCTP chimera; reduced circulatory survival compared to the WT subunit (that has the natural CTP) bwt wt 1000 bboCTP v5 b117 117 100 10 1 0 10 20 30 40 50 60 70 80 90 100 Time (min) Parameter bwt bboCTP b117 C0 (ng/ml) 875 ± 200a 265 ± 40b 1560±250b 185 ± 20b 970 ± 80b 24.6 ± 0.7b 17.6 ± 1.0c AUC (ng.min/ml) t1/2 (min) Gabay et al., 2014 8125 ± 1360a 47.2 ± 1.8a (different letters P<0.01) Association of the CGb variants with the human a subunit in transfected CHO cells to form heterodimers; Conformation-sensitive epitopes on heterodimers and monomeric subunit variants A C Heterodimer assembly mAb INN-53 Heat: MW (kDa) 90 49 - - + - + + - + Heterodimer 35 B Heterodimeric-like conformation hCGα hCGβ INN-53 (bL2 & aL1) INN-68 (uncombined b near Cys knot) b Subunit 26 19 1 D 2 3 4 5 6 7 8 mAb INN-68 MW (kDa) 117 90 Heterodimer 49 b Subunit 35 26 19 1 Gabay et al., 2014 2 3 4 b mono. 5 6 a/b di. 7 Progesterone (pg/ml) Bioactivity of the of the CGboCTP heterodimer; immortalyzed rat granulosa cell bioassay 2000 1500 1000 500 0 0.01 0.1 1 10 100 Heterodimer (ng/ml) Gabay et al., 2014 Heterodimer Max. Progesetrone (pg/ml) EC50 (ng/ml) CGwt 1515 ± 210 1.5 ± 0.5 CGboCTP 1555 ± 205 1.5 ± 0.4 CG117 1570 ± 255 1 ± 0.3 1000 How the intracellular behavior of the equine LH/CGb subunits fulfill the needs for biosynthesis both in the pituitary and placenta? • A single gene encodes the LHb and CGb subunits in equids in these two organs (known in the horse as eLH/CGb; no CTP lacking lutropic subunit) • Together with the a subunit, the eLH/CGb gene is expressed in the pituitary to synthesize eLH and in the placenta to produce eCG (also known as PMSG) as part of reproduction endocrinology in mares • The pituitary eLHb and placental eCGb subunits share the same amino acid composition and both have a O-glycosylated CTP Whether the secretion kinetics and routing of the eLH/CGb subunit from transfected cells are strictly hLHb- or hCGb-like, or combines characteristics of both? Differences in the intracellular behavior of the human LHb and CGb subunits • In primates, the LHb and CGb subunits are products of different genes which are efficiently expressed in the gonadotropes and trophoblasts, respectively • Despite the similarities between the human LH and CG b subunits, the storage and secretion profiles of the heterodimers differ. Whereas The secretion of the hLHb subunit is slow and inefficient, that of the hCGb subunit is fast and quantitative • Differences in the secretion from MDCK cells (hLHb- basolateral; hCGb apical) Whether the secretion kinetics and routing of the eLH/CGb subunit from transfected cells are strictly hLHb- or hCGb-like, or combines characteristics of both? Expression and secretion of the eLH/CGβ, hCGβ and LHβ subunits in transfected CHO cells A Mw (kDa) B eLH/CGb L Mw (kDa) 117 90 49 M L M L M 117 90 49 C hCGb L M Mw (kDa) L M L M eLH/ L M 47 N2 N1 26 26 19 L M 118 85 35 35 LHb 19 36 26 CGb 20 IP: 1 2 antieLH/CG Media Recovery (%): 3 4 5 6 anti- NRS hCGb IP: 25.6 ± 7.0 MDCK 17.3 ± 4.4 1 2 antihCGb 5 6 3 4 anti NRS eLH/CG 82.6 ± 6.0 MDCK 81.6 ± 5.5 Kinetics: t1/2 (hr) = 6.6 ± 0.2 t1/2 (hr) = 1.5 ± 0.2 (Pulse chase) Recovery (%) = 16 ± 2 Recovery (%) = 63 ± 4 LHb 1 2 human 3 4 bovine <10% Cohen et al., 2015 Apical secretion of the eLH/CGβ and hCGβ subunits from polarized MDCK cells A B hCGb Mw Apical (kDa) Basolateral Apical eLH/CGb Basolateral 100 100 100 80 80 80 117 90 49 60 36 40 26 19 20 1 IP: 2 3 4 5 6 7 8 0 Percent of total secretion eLH/CGb ** 66% hCGb ** 65% 60 60 35% 34% 40 40 20 20 0 0 Ap BL Ap BL Cohen et al., 2015 Summary (a): A role of the Carboxy-Terminal-Peptide Oglycosylation in the LHb to CGb evolution •The LHb to CGb gene conversion is potentially wide-spread •When translated, the cryptic boCTP stretch does not prevent crucial aspects of hormone biosynthesis (the assembly of the heterodimer, formation of conformational-sensitive epitopes and the activation of the cognate receptor). However, this domain is missing the set of Olinked glycans and lacks the hallmark function of prolonging the circulatory survival and determinants for apical secretion which are typical to the naturally expressed O-glycosylated CTP domain •The absence of extensive O-glycosylation and the associated failure to gain new hormonal properties provides an explanation as to why LH did not evolve into CG in ruminants, and possibly in additional species, that apply different strategies to delay luteolysis at the early stages of gestation Summary (b): The production of the LH/CGb subunit in equids • The equine LH/CGb subunit combines intracellular traits that diverged in the case of the human LHb and CGb subunits • We propose that the distinguished intracellular behavior of the equine gonadotropin subunit evolved in association with the needs for biosynthesis in the pituitary and placenta Acknowledgments: Sigal Nakav, Shelly Kaner and Reut Gabay Albena Samokovlisky, Yehudit Amor and Rakefet Rosenfeld Ed Grotjan and Prabhjit Chadna-Mohanty Irv Boime and Albina Jablonka-Shariff Riad Agbaria, Mazal Rubin, Zvi Ben-Zvi and David Stepensky Peter Berger Fortune Kohen and Abraham Amsterdam Limor Cohen George Bousfield