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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