O-glycosylation and protein evolution: the case of the LH to CG development David Ben-Menahem Clinical Biochemistry and Pharmacology, Faculty of Health.

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

David Ben-MenahemClinical Biochemistry and Pharmacology ,

Faculty of Health SciencesBen-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

LH COOHNH2 301 121

hCGo o o o

COOHNH2 30131 145

COOHNH2 521 9278α

FSH COOHNH2 71 110

Primates/Equids

24

CTP

The LH to CG subunit development; Carboxy Terminal Peptide extention (CTP) characteristics

• 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 hCG from the apical side of placental trophoblasts into the maternal circulation to delay luteolysis in primates

hLH COOHNH2 301 121

o o o o COOHhCG NH2 30131 145

CTP

FS

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

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

bLHβ: 110 CDHPPLPDILFL121

bLHβboCTP: 110CD....P…QTSSSSKDAPLQP...PMPILTLQTSRHSS PPFPIKTS147

eLH/CGβ: 110CA....P…QASSSSKDPPSQPLTSTSTPTPGASRRSSHPLPIKTS149

hCGβ: 110CDDPRFQASSSSKAPPP...SLPSPSRL...PGPSDTPILPQ145

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?

Nakav et al., 2005

LHβCTP1421 111

LHβ huCTP

1

LHβ

121

+ huCTP

1

LHβ121

LHβboCTP1

boCTP

147111

LHβ

FS

LHβ

1 111

LHβ LHβ111

1211

Expression and secretion of the bovine elongated LHβboCTP subunit in transfected CHO cells

Nakav et al., 2005

CG

hLH: 110CDHPQLSGLLFL121

hCGwt: 110CDDPRFQASSSSKAPPPSLPSPSRLPGPSDTPILPQ145

hCGboCTP (boCTP) 110CDDPRFQASSSSKDAPLQPPMPILTLQTSRHSSPPFPIKTS150

hCG117 (117) 110CDDPRFQA117

Structure and Function of the boCTP Domain

1 145117hCG CTP hCG boCTP

1 150 1 117hCG

wt boCTP 117

Cloned in PM2 and stably transfected into CHO cells

Secretion kinetics: Pulse Chase analysis

Recovery :(%)

t½ (min) :

65 ± 5 65 ± 5

80 ± 5

50 ± 5

90 ± 5115 ± 10

Nakav et al., 2005

Lectin array analysis of the secreted chimeric subunit; absence of mucin type O-glycans

o o o o

CTP

CGβWT

boCTP

?

CGβboCTP CGβ117F

luor

esce

nce

(A.U

.)

Gabay et al., 2014

Basolateral secretion of the CGboCTP chimera from polarized MDCK cells

30 hCG boCTP

CGCGboCTPboCTP

7070

Basolateral)%( Apical(%)

Nakav et al., 2005

20 8080

757525

356565

CG- Odg

LH

CG

301 121

13 145CTP

o o o o

301

30131 145CTP

Basolateral(%) Apical(%) Boime and his colleagues

Pharmacokinetics of the CGβboCTP chimera; reduced circulatory survival compared to the WT subunit (that has the natural CTP)

1

10

100

1000

0 10 20 30 40 50 60 70 80 90 100

Time (min)

sub

unit

(ng/

ml)

wt

v5

117

wt

boCTP

117

t1/2 (min) 47.2 ± 1.8a 24.6 ± 0.7b 17.6 ± 1.0c

AUC (ng.min/ml) 8125 ± 1360a 1560±250b 970 ± 80b

C0 (ng/ml) 875 ± 200a 265 ± 40b 185 ± 20b

Parameter wt boCTP 117

)P

lasm

a C

once

ntra

tion

(ng/

ml)

(different letters P<0.01) Gabay et al., 2014

49

35

26

19

90

- + - +

- +

- +

hCG CGwt CGboCTPCG117

Heat:

1 2 3 4 5 6 7 8

Heterodimer

C mAb INN-53

Subunit

MW (kDa)

49

35

26

19

90117

MW (kDa)

Heterodimer

Subunit

1 2 3 4 5 6 7 mono. / di.

hCG CGwt CG117

wt boCTP117

CGboCTP

D mAb INN-68

Heterodimeric-like conformation B

hCGβ

hCGα

INN-68 (uncombined near Cys knot)

INN-53 (L2 & L1)

Heterodimer assembly

Association of the CG variants with the human subunit in transfected CHO cells to form heterodimers; Conformation-sensitive epitopes on heterodimers and monomeric subunit variants

A

Gabay et al., 2014

0

500

1000

1500

2000

0.01 0.1 1 10 100 1000

Heterodimer (ng/ml)

Prog

este

rone

(pg

/ml)

Bioactivity of the of the CGboCTP heterodimer; immortalyzed rat granulosa cell bioassay

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

Gabay et al., 2014

• A single gene encodes the LH and CG subunits in equids in these two organs (known in the horse as eLH/CG; no CTP lacking lutropic subunit)

• Together with the subunit, the eLH/CG 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 eLH and placental eCG subunits share the same amino acid composition and both have a O-glycosylated CTP

Whether the secretion kinetics and routing of the eLH/CG subunit from transfected cells are strictly hLH- or hCG-like, or

combines characteristics of both?

How the intracellular behavior of the equine LH/CG subunits fulfill the needs for biosynthesis both in the pituitary and placenta?

• 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 hLH subunit is slow and inefficient, that of the hCG subunit is fast and quantitative

• Differences in the secretion from MDCK cells (hLH- basolateral; hCG apical)

Differences in the intracellular behavior of the human LH and CG subunits

Whether the secretion kinetics and routing of the eLH/CG subunit from transfected cells are strictly hLH- or hCG-like, or combines

characteristics of both?

A eLH/CG

Mw(kDa)

B hCG

117

4990

35

26

19

Mw(kDa)

1 2 3

IP:

4 5anti -

eLH/CG

6anti-

hCGNRS

L LLM MM

N2N1

118

47

85

36

26

20

Mw(kDa) L M L M

1 2 3 4human bovine

LH

eLH/

CG

117

49

90

35

2619

L LLM MM

1 2 3

IP:

4 5anti-

eLH/CG

6anti-

hCGNRS

C LH

Media Recovery :(%)

25.6 ± 7.0 82.6 ± 6.0 <10%

Expression and secretion of the eLH/CGβ, hCGβ and LHβ subunits in transfected CHO cells

MDCK 17.3 ± 4.4 MDCK 81.6 ± 5.5

t1/2 (hr) = 6.6 ± 0.2 t1/2 (hr) = 1.5 ± 0.2

Recovery (%) = 16 ± 2 Recovery (%) = 63 ± 4

Kinetics: (Pulse chase)

Cohen et al., 2015

eLH/CG

Apical Basolateral

1 2 3 4

IP:an

ti -

eLH/C

G

NRS

NRS

anti

-

eLH/C

G

117

49

90

36

26

19

Mw(kDa)

hCG

Apical Basolateralan

ti -

hCG NRS

NRS

5 6 7 8

anti

-hC

G

Per

cen

t of

tot

al s

ecre

tion

eLH/CG hCG

34%

65%66%

35%

Ap ApBL BL

** **

A B

Apical secretion of the eLH/CGβ and hCGβ subunits from polarized MDCK cells

Cohen et al., 2015

•The LH to CG 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

Summary (a): A role of the Carboxy-Terminal-Peptide O-glycosylation in the LH to CG evolution

• 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

Summary (b): The production of the LH/CG subunit in equids

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

Acknowledgments:

Limor Cohen George Bousfield