Transcript שקופית 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
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