O-glycosylation and protein evolution the case of the

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 NH 2 1 FSH NH 2 1 LH NH 2 1 52 7 24 30 Primates/Equids h. CG NH 2 1 13 30 78 92 COOH 110 COOH 121 COOH o o COOH 145 CTP

The LH to CG subunit development; Carboxy Terminal Peptide extention (CTP) characteristics FS h. LH NH 2 1 30 121 COOH o o h. CG NH 2 1 13 145 COOH 30 CTP • The CG gene presumably evolved from the ancestral LH gene • Ser/Thr/Pro rich domain, multiple O-glycans attached to the CTP (4 -12) • Prolongs circulatory survival compared to LH • Orient secretion of h. CG 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 LH gene is conserved among mammals, few mutations localized to a small region and the gain of new hormonal properties Whether the LH genes in species other than primates and equids contain an untranslated CTP-like sequence? Yes, a CTP-like sequence is cryptic in the LH gene of several mammals but not in birds, amphibians and fishes Whether the incorporation of the cryptic CTP sequence in the bovine LH reading frame will result in misfolding and degradation or allow the expression of the extended subunit? b. LHβ: 110 CDHPPLPDILFL 121 b. LHβbo. CTP: 110 CD. . P…QTSSSSKDAPLQP. . . PMPILTLQTSRHSS PPFPIKTS 147 e. LH/CGβ: 110 CA. . P…QASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTS 149 h. CGβ: 110 CDDPRFQASSSSKAPPP. . . SLPSPSRL. . . PGPSDTPILPQ 145 Nakav et al. , 2005

Expression and secretion of the bovine elongated LHβbo. CTP subunit in transfected CHO cells FS 1 111 1 121 LHβ 1 bo. CTP 121 LHβ + D 121 1 LHβ Nakav et al. , 2005 LHβ 147 LHβ 111 1 LHβ 142 111 1 hu. CTP LHβbo. CTP LHβ 111 hu. CTP LHβCTP

Structure and Function of the bo. CTP Domain h. LH : 110 CDHPQLSGLLFL 121 h. CG ( wt): 110 CDDPRFQASSSSKAPPPSLPSPSRLPGPSDTPILPQ 145 h. CG bo. CTP ( bo. CTP) 110 CDDPRFQASSSSKDAPLQPPMPILTLQTSRHSSPPFPIKTS 150 CGb h. CG 117 ( 117) 110 CDDPRFQA 117 Cloned in PM 2 and stably transfected into CHO cells Secretion kinetics: Pulse Chase analysis 1 117 h. CG b wt Recovery (%): t½ (min): Nakav et al. , 2005 65 ± 5 80 ± 5 145 CTP 1 h. CG 150 bo. CTP bbo. CTP 65 ± 5 115 ± 10 1 117 h. CG b 117 50 ± 5 90 ± 5

Lectin array analysis of the secreted chimeric subunit; absence of mucin type O-glycans CGβbo. CTP CGβWT o o CTP CGβ 117 ? Fluorescence (A. U. ) bo. CTP N-glycans Gabay et al. , 2014 O-glycans

Basolateral secretion of the CG bo. CTP chimera from polarized MDCK cells Apical (%) CGbbo. CTP h. CG 30 70 bo. CTP Boime and his colleagues Apical (%) Basolateral (%) CGb 65 1 13 30 CTP 35 25 o o 75 145 LHb 1 30 121 CGb - Odg 20 1 13 30 CTP 80 145 Nakav et al. , 2005

Pharmacokinetics of the CGβbo. CTP chimera; reduced circulatory survival compared to the WT subunit (that has the natural CTP) ) Plasma Concentration (ng/ml) wt bo. CTP 117 Parameter bwt bbo. CTP b 117 C 0 (ng/ml) 875 ± 200 a 265 ± 40 b 1560± 250 b 185 ± 20 b 970 ± 80 b 24. 6 ± 0. 7 b 17. 6 ± 1. 0 c AUC (ng. min/ml) t 1/2 (min) Gabay et al. , 2014 8125 ± 1360 a 47. 2 ± 1. 8 a (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 m. Ab INN-53 Heterodimer assembly G h. C Heat: MW (k. Da) 90 49 - + wt CG - + TP C Gbo C - + 117 CG - + Heterodimer 35 B Heterodimeric-like conformation h. CGα h. CGβ INN-53 (b. L 2 & a. L 1) INN-68 (uncombined b near Cys knot) Subunit 26 19 1 2 3 4 5 6 7 8 D m. Ab INN-68 h. CG P TP t o. CT 17 wt bo. C 117 CGw CGb CG 1 MW (k. Da) 117 90 49 Heterodimer 35 Subunit 26 19 1 2 3 4 5 6 7 Gabay et al. , 2014 mono. a/ di.

Bioactivity of the CGbo. CTP heterodimer; immortalyzed rat granulosa cell bioassay Heterodimer EC 50 (ng/ml) CGwt 1515 ± 210 1. 5 ± 0. 5 CGbo. CTP 1555 ± 205 1. 5 ± 0. 4 CG 117 Gabay et al. , 2014 Max. Progesetrone (pg/ml) 1570 ± 255 1 ± 0. 3

How the intracellular behavior of the equine LH/CG subunits fulfill the needs for biosynthesis both in the pituitary and placenta? • A single gene encodes the LH and CG subunits in equids in these two organs (known in the horse as e. LH/CG ; no CTP lacking lutropic subunit) • Together with the a subunit, the e. LH/CG gene is expressed in the pituitary to synthesize e. LH and in the placenta to produce e. CG (also known as PMSG) as part of reproduction endocrinology in mares • The pituitary e. LH and placental e. CG subunits share the same amino acid composition and both have a O-glycosylated CTP Whether the secretion kinetics and routing of the e. LH/CG subunit from transfected cells are strictly h. LH - or h. CG -like, or combines characteristics of both?

Differences in the intracellular behavior of the human LH and CG subunits • In primates, the LH and CG 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 subunits, the storage and secretion profiles of the heterodimers differ. Whereas The secretion of the h. LH subunit is slow and inefficient, that of the h. CG subunit is fast and quantitative • Differences in the secretion from MDCK cells (h. LH - basolateral; h. CG apical) Whether the secretion kinetics and routing of the e. LH/CG subunit from transfected cells are strictly h. LH - or h. CG -like, or combines characteristics of both?

Expression and secretion of the e. LH/CGβ, h. CGβ and LHβ subunits in transfected CHO cells A e. LH/CG Mw (k. Da) L C LH B h. CG Mw (k. Da) 117 90 49 M L M 117 90 49 L M L M e. LH/ CG L M 47 N 2 N 1 26 26 19 L M 118 85 35 35 Mw (k. Da) 19 36 26 20 IP: 1 2 3 4 5 6 anti- NRS e. LH/CG h. CG Media Recovery (%): IP: 25. 6 ± 7. 0 MDCK 17. 3 ± 4. 4 1 2 anti h. CG 5 6 3 4 anti NRS e. LH/CG 82. 6 ± 6. 0 MDCK 81. 6 ± 5. 5 Kinetics: t 1/2 (hr) = 6. 6 ± 0. 2 Recovery (%) = 16 ± 2 Recovery (%) = 63 ± 4 1 2 human 3 4 bovine <10% t 1/2 (hr) = 1. 5 ± 0. 2 (Pulse chase) LH Cohen et al. , 2015

Apical secretion of the e. LH/CGβ and h. CGβ subunits from polarized MDCK cells A B h. CG Mw Apical (k. Da) e. LH/CG 117 90 49 36 26 19 RS 8 ** 66% ** 65% 34% Ap BL N 7 6 a h. C nti G N RS 5 N RS 4 RS 3 e. L anti H /C G 2 N IP: e. L anti H - /C G 1 h. CG Basolateral Apical Percent of total secretion e. LH/CG Cohen et al. , 2015

Summary (a): A role of the Carboxy-Terminal-Peptide Oglycosylation in the LH to CG evolution • The LH to CG gene conversion is potentially wide-spread • When translated, the cryptic bo. CTP 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/CG subunit in equids • The equine LH/CG subunit combines intracellular traits that diverged in the case of the human LH and CG 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