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O-glycosylation and protein evolution: the case of the LH b to CG b development

This article explores the evolution and function of O-glycosylation in the LHb to CGb subunit development, specifically focusing on the Carboxy Terminal Peptide extension (CTP).

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O-glycosylation and protein evolution: the case of the LH b to CG b development

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  1. 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

  2. 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)

  3. The Gonadotropin Subunits COOH α NH2 1 92 52 78 FSH NH2 COOH 110 1 24 7 LH NH2 COOH 1 30 121 Primates/Equids o o o o COOH hCG NH2 1 30 145 13 CTP

  4. COOH hLH NH2 1 30 121 The LHb to CGb subunit development; Carboxy Terminal Peptide extention (CTP) characteristics FS o o o o hCG NH2 COOH 1 30 145 13 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

  5. 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...PMPILTLQTSRHSSPPFPIKTS147 eLH/CGβ: 110CA....P…QASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTS149 hCGβ: 110CDDPRFQASSSSKAPPP...SLPSPSRL...PGPSDTPILPQ145 Nakav et al., 2005

  6. Expression and secretion of the bovine elongated LHβboCTP subunit in transfected CHO cells FS 1 111 147 1 121 LHβboCTP LHβ LHβ boCTP 142 111 1 LHβCTP 1 121 LHβ huCTP + LHβ huCTP 1 D 121 LHβ 111 1 LHβ111 LHβ Nakav et al., 2005

  7. 1 1 1 117 117 145 150 hCGb hCGb hCGb boCTP CTP Structure and Function of the boCTPDomain hLHb: 110CDHPQLSGLLFL121 hCGb (bwt): 110CDDPRFQASSSSKAPPPSLPSPSRLPGPSDTPILPQ145 hCGbboCTP (bboCTP) 110CDDPRFQASSSSKDAPLQPPMPILTLQTSRHSSPPFPIKTS150 hCGb117 (b117) 110CDDPRFQA117 CGb Cloned in PM2 and stably transfected into CHO cells Secretion kinetics: Pulse Chase analysis b wt b117 bboCTP Recovery (%): 65 ± 5 65 ± 5 50 ± 5 80 ± 5 115 ± 10 90 ± 5 t½ (min): Nakav et al., 2005

  8. CGβboCTP CGβ117 CGβWT ? o o o o CTP boCTP O-glycans N-glycans Lectinarray analysis of the secreted chimeric subunit; absence of mucin type O-glycans Fluorescence (A.U.) Gabay et al., 2014

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

  10. Pharmacokinetics of the CGβboCTP chimera; reduced circulatory survival compared to the WT subunit (that has the natural CTP) bwt bboCTP b117 ) Plasma Concentration (ng/ml) Parameter bwt bboCTP b117 C0 (ng/ml) 875 ± 200a 265 ± 40b 185 ± 20b AUC (ng.min/ml) 1560±250b 970 ± 80b 8125 ± 1360a t1/2 (min) 47.2 ± 1.8a 24.6 ± 0.7b 17.6 ± 1.0c Gabay et al., 2014 (different letters P<0.01)

  11. 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 CGboCTP 90 CG117 hCG CGwt Heat: 49 35 26 Heterodimer - + - + - + - + 19 1 2 3 4 5 6 7 8 hCGα hCGβ CmAb INN-53 A Heterodimer assembly MW (kDa) MW (kDa) 117 B Heterodimeric-like conformation 90 DmAb INN-68 bSubunit 49 Heterodimer bboCTP b117 CG117 CGboCTP CGwt bwt hCG 35 bSubunit 26 19 1 2 3 4 5 6 7 a/b di. bmono. Gabay et al., 2014 INN-68 (uncombined b near Cys knot) INN-53 (bL2 & aL1)

  12. Bioactivity of the of the CGboCTP heterodimer; immortalyzed rat granulosa cell bioassay Gabay et al., 2014

  13. 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?

  14. 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?

  15. B hCGb Expression and secretion of the eLH/CGβ,hCGβand LHβsubunits in transfected CHO cells Mw (kDa) Mw L M L M L M M L L L M M L M L M ( kDa ) 117 90 117 118 90 85 49 49 47 35 N2 N1 26 35 36 eLH/ CGb 26 19 26 19 LHb 20 3 1 2 4 6 5 1 2 4 6 3 5 1 2 3 4 anti- eLH/CG anti- hCGb NRS NRS anti - eLH/CG human bovine IP: anti- hCGb IP: C LHb A eLH/CGb Mw (kDa) Media Recovery (%): 25.6 ± 7.0 82.6 ± 6.0 <10% MDCK 17.3 ± 4.4 MDCK 81.6 ± 5.5 t1/2 (hr) = 1.5 ± 0.2 t1/2 (hr) = 6.6 ± 0.2 Kinetics:(Pulse chase) Recovery (%) = 63 ± 4 Recovery (%) = 16 ± 2 Cohen et al., 2015

  16. hCGb eLH/CGb hCGb eLH/CGb Mw (kDa) Basolateral Apical Apical Basolateral ** ** 117 66% 65% 90 49 36 35% 34% 26 19 7 1 2 3 4 6 5 8 anti -hCGb anti -hCGb anti - eLH/CG anti -eLH/CG IP: NRS NRS NRS NRS Ap BL Ap BL Percent of total secretion Apical secretion of the eLH/CGβ andhCGβ subunits from polarized MDCK cells A B Cohen et al., 2015

  17. Summary (a): A role of the Carboxy-Terminal-Peptide O-glycosylation 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 O-linked 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

  18. 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

  19. Acknowledgments: Sigal Nakav, Shelly Kaner and Reut Gabay Albena Samokovlisky, Yehudit Amor and Rakefet RosenfeldEd Grotjan and Prabhjit Chadna-Mohanty Irv Boime and Albina Jablonka-ShariffRiad Agbaria, Mazal Rubin, Zvi Ben-Zvi and David Stepensky Peter BergerFortune Kohen and Abraham Amsterdam Limor Cohen George Bousfield

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