Download Hamster Placental Lactogen-ll Contains a Structural Feature Unique

Hamster Placental Lactogen-ll
Contains a Structural Feature Unique
among the Growth HormoneProlactin-Placental Lactogen Family
Jonathan N. Southard, Luan Do*, William C. Smith, and
Frank Talamantes
Department of Biology
University of California
Santa Cruz, California 95064
Sequence analysis of cDNA for hamster placental
lactogen-ll (PL-II) revealed that while this protein
has a high degree of sequence homology to mouse
and rat PL-II it contains a pair of cysteine residues
not present in the mouse and rat proteins or in any
other known member of the GH-PRL-PL protein family. This unique pair of cysteine residues may be
responsible for the extreme tendency of hamster
PL-II, compared to other members of the GH-PRLPL family, to form disulfide-bonded hormone-serum
protein complexes. (Molecular Endocrinology 3:
1710-1713, 1989)
ior of haPL-ll might be a reflection of a unique structural
feature of this member of the GH-PRL-PL hormone
family. The cDNA-deduced amino acid sequence of
haPL-ll was determined in order to examine this possibility.
RESULTS AND DISCUSSION
To obtain cDNAs for haPL-ll, a A expression library was
constructed using cDNA synthesized from RNA of day16 pregnant hamster placenta. Five positive clones
were obtained from immunoscreening of 1 x 105 clones
using a polyclonal antiserum to haPL-ll (6). Two clones
with cDNAs of approximately 800 base pairs (bp) were
sequenced. Subsequent screening of the library was
performed by hybridization to a 174 bp fragment from
the 5'-end of one of these cDNAs. One of the clones
obtained by hybridization had a cDNA of approximately
900 bp which was also sequenced. The composite
nucleotide sequence for the three cDNAs is shown in
Fig. 1. The overlapping portions of the cDNAs differed
only in the starting point for the poly(A) tail; it follows
nucleotide 850 for two of the cDNAs and nucleotide
857 for the third. The composite sequence contains an
open reading frame for a 221 amino acid polypeptide.
Amino terminal amino acid sequencing of purified
haPL-ll demonstrated that the amino terminus of the
mature haPL-ll protein is located 31 amino acids from
the amino terminal Met of the cDNA-deduced sequence
(Fig. 1). The mRNA for haPL-ll therefore codes for a
221 amino acid precursor protein which is cleaved to
yield a 191 amino acid mature haPL-ll protein. Similarly,
mPL-ll and rPL-ll are synthesized as 222 amino acid
precursors which are cleaved to yield mature proteins
of 191 amino acid residues (4, 5). As is the case for
mPL-ll and rPL-ll, the mature haPL-ll protein does not
contain a consensus sequence for Asn-linked glycosylation (Asn-X-Ser/Thr).
Figure 2 shows the alignment of the predicted amino
acid sequences of haPL-ll, mPL-ll, and rPL-ll. A high
INTRODUCTION
Placental lactogen II (PL-II), a member of the GH-PRLPL family of structurally related hormones, has been
purified from three rodent species: mouse (mPL-ll), rat
(rPL-ll), and hamster (haPL-ll) (1-3). Complementary
DNAs for mPL-ll and rPL-ll have been sequenced (4, 5)
and the deduced amino acid sequences have 79%
sequence identity. The three purified PL-lls have similar
Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyclonal antiserum to
each PL-II cross-reacts with the heterologous PL-lls
under both native and denaturing conditions (6). The
structures of the PL-lls therefore appear to be generally
similar. In at least one respect, however, the behavior
of haPL-ll differs markedly from that of mPL-ll and rPLII. In both the placenta and the maternal circulation,
haPL-ll is present primarily as high Mr (Mr > 100,000)
disulfide-bonded forms (3, 7), while mPL-ll and rPL-ll
are present primarily in monomeric form (8, 9). Recent
evidence (10) indicates that the major circulating form
of haPL-ll is a disulfide-bonded complex of haPL-ll and
a serum glycoprotein, possibly a-2-macroglobulin. It
seemed likely that the unusual disulfide-bonding behav0888-8809/89/1710-1713$02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
1710
Hamster PL-II cDNA
1711
AGCGGCTTTCCTCTGTTGTAGTCCACAGTGCAACACATCTTCTCAGAG
-30
-20
4 9 ATG CAG CTG CCT TTG ACT CCA CTG TCC TTC TCT GGG ACA CTC CTT TTG ATG GCA ATG TCA
MET Gin Leu Pro Leu Thr Pro Leu Ser Phe Ser Gly Thr Leu Leu Leu MET Ala MET Ser
-10
-1 +1
109 AAT TTT CTC CTT TGG GAA CAT GTG ACC TCC TCA GCA AGT CCT CGT TTA TCC ACT AGA AAC
Asn Phe Leu Leu Trp Glu His Val Thr Ser Ser Ala Ser Pro Arg Leu Ser Thr Arg Asn
11
21
169 TTG TAC CAG CGT GTG GTT GAA TTG TCA CAC TGC ACC CAT GAT CTT GCC TCA AAA GTT TTC
Leu Tyr Gin Arg Val Val Glu Leu Ser His Cys Thr His Asp Leu Ala Ser Lys Val Phe
31
41
22 9 ACT GAC TTT AAT ATG AAG TTT GGT AAG AGT ATT TGC AGA CAG AAA CTG ATG TTA TAC ACC
Thr Asp Phe Asn MET Lys Phe Gly Lys Ser lie Cys Arg Gin Lys Leu MET Leu Tyr Thr
51
61
28 9 TGC CAC ACC TCC TCT ATT CCT ACT CCA GAA AAC AGA GAG CAA GTC CAC CAA ACA AAC TCG
Cys His Thr Ser Ser lie Pro Thr Pro Glu Asn Arg Glu Gin Val His Gin Thr Asn Ser
71
81
34 9 GAA GAT CTC CTG AAA GTG ACG ATC AGT GTT TTA CAA GCC TGG GAG GAG CCT GTG AAG CAC
Glu Asp Leu Leu Lys Val Thr He Ser Val Leu Gin Ala Trp Glu Glu Pro Val Lys His
91
101
4 09 ATG GTG GCT GCA GTA GCT GCT CTT CCA GGT ACA TCT GAT GCC ATG CTG TCA AGA GCA AAA
MET Val Ala Ala Val Ala Ala Leu Pro Gly Thr Ser Asp Ala MET Leu Ser Arg Ala Lys
111
121
4 69 GAG TTG GAG GAA AGA GTT TTA GGC CTT CTG GAG GGA CTG AAG ATC ATA CTC AAC AGG ATT
Glu Leu Glu Glu Arg Val Leu Gly Leu Leu Glu Gly Leu Lys He He Leu Asn Arg He
131
141
52 9 CAT CCT GGA GCT GTT GAA AAT GAC TAT ACT TTC TGG TCT GGA TGG TCA GAT TTG CAG TCA
His Pro Gly Ala Val Glu Asn Asp Tyr Thr Phe Trp Ser Gly Trp Ser Asp Leu Gin Ser
151
161
58 9 TCT GAT GAA GCT ACT CGT AAC ATT GCT TTT TAT ACT ATG GGC CGT TGC CTG CGC AGG GAT
Ser Asp Glu Ala Thr Arg Asn He Ala Phe Tyr Thr MET Gly Arg Cys Leu Arg Arg Asp
171
181
64 9 ACA CAC AAA GTT GAT AAT TAT CTC AAG GTT TTG AAA TGC CGA GAT ATC CAT AAT AAC AAC
Thr His Lys Val Asp Asn Tyr Leu Lys Val Leu Lys Cys Arg Asp He His Asn Asn Asn
191
709 TGC TGA
Cys *
786
GCTCAAATCCTTAACCACTGTCATGGAGAAGGTCCAGACCTCAAAGTTCCATTGAGTCTTTACCTTTTGGT
TCATTCCTTGGTTTAATGGGCATGTTATTCAAAAATAAACATTGATTCTTTGAAATGCTTAATTCAAAATGAAAAAAAA
8 65 AAAAAAAAAAAAAAAAAAAAAAAAA
Fig. 1. Composite Nucleotide Sequence and Predicted Amino Acid Sequence for haPL-ll cDNA
The amino acid residues determined by amino terminal sequence analysis of haPL-ll are underlined. The three cDNAs sequenced
begin at nucleotides 1,91, and 100 and extend to the poly(A) tail. The overlapping portions of the three cDNAs differ only in the
starting point for the poly(A) tail (see text).
degree of sequence homology is apparent for the three
proteins. Most notable are two regions, one of 30 amino
acids (residues 70-99) and another of 27 amino acids
(165-191), in which there are no nonconservative substitutions in haPL-ll relative to mPL-ll and only three in
each case relative to rPL-ll. Overall, haPL-ll has essentially identical sequence homology to both mPL-ll (70%
identity) and rPL-ll (68% identity). This homology is
slightly less than that between mPL-ll and rPL-ll (79%
identity).
The GH-PRL-PL protein family comprises pituitary
GH and PRL and several placentally derived proteins.
Production of two PLs, designated PL-I and PL-II, appears to be common in rodents. In addition, several
cDNAs have been obtained from the placenta which
code for other PRL-like proteins (11). Although high Mr
forms have been detected for several members of the
GH-PRL-PL family, it appears that these proteins circulate in the blood primarily as monomers. The only
known exception is haPL-ll. The predominant form of
haPL-ll in the maternal circulation is a species with a
Mr of 600,000 as determined by gel filtration and
360,000 as determined by SDS-PAGE. Monomeric
haPL-ll can be released from this high Mr form by
reduction of disulfide bonds (10). While this high Mr
form of haPL-ll has not been completely characterized,
it was demonstrated that purified (monomeric) haPL-ll
readily forms a similar high Mr species when incubated
Vol 3 No. 11
MOL ENDO-1989
1712
haPL-II
mPL-II
rPL-II
-20
M Q L P L T P L S F S G T L L L M A M S N F L L W E H V T
M K L S L S Q P C S F S G A L L L L A V S N L L V W E K V T
M V Q L S L T Q P C F S G T L L M L A V S T L L L W E Q V T
haPL-II
mPL-II
rPL-II
20 *
1
S S A S P R L S T R N L Y Q R V V E L S H C T H D L A S K V
S L P N Y R L P T E S L Y Q R V I V V S H N A H D L A S K A
S A P N Y R M S T G S L Y Q R V V E L S H Y T H D L A S K V
haPL-II
mPL-II
rPL-II
40
*
F T D F N M K F G K S I C R Q K L M L Y T C H T S S
I P T P
F M E F E M K F G R T A W T Y G L M L S P C H T A A I L T P
F I E F D M K F G R T V W T H N L M L S P C H T A A I P T P
haPL-II
mPL-II
rPL-II
80
E N R E Q V H Q T N S E D L L K V T I S V L Q A W E E P V K
E N S E Q V H Q T T S E D L L K V S I T I L Q A W E E P L K
E N S E Q V H Q A K S E D L L K V S I T I L Q A W Q E P L K
haPL-II
mPL-II
rPL-II
100
H M V A A V A A L P G T S D A M L S R A K E L E E R V L G L
H M V A A V A A L P H V P D T L L S R T K E L E E R I Q G L
H I V A A V A T L P D G S D T L L S R T K E L E E R I Q G L
60
haPL-II
mPL-II
rPL-II
haPL-II
mPL-II
rPL-II
haPL-II
mPL-II
rPL-II
120
140
L E G L K I
I L N R I H P G A V E N D Y T F W S G W S D L Q
L E G L K I
I F N R V Y P G A V A S D Y T F W S A W S D L Q
L E G L E T I L S R V Q P G A V G S D Y T F W S E W S D L Q
160
S S D E A T R N I A F Y T M G R C L R R D T H K V D N Y L K
S S D E S T K N S A L R T L W R C V R R D T H K V D N Y L K
S S D K S T K N G V L S V L Y R C M R R D T H K V D N F L K
180
V
V
V
'
L K C R D I H N N N C
L K C R D V H N N N C
L K C R D I Y N N N C
Fig. 2. Alignment of the Predicted Amino Acid Sequences of haPL-II, mPL-II, and rPL-II
Nonconservative substitutions are indicated by boldface type (conservative substitutions: D = E, N = Q, K = R, S = T, I = L =
V). The asterisks indicate the unique cysteine residues of haPL-II. The sequence for mPL-II is from Jackson et al. (4) and that for
rPL-II is from Duckworth et al. (5).
with hamster serum in vitro (10). This suggested that
the major circulating form of haPL-II is a disulfidebonded complex between haPL-II and a serum protein.
The predicted amino acid sequence of haPL-II confirms
this. While the sequence does not contain a site for
Asn-linked glycosylation, the major circulating form of
haPL-II is about 10% Asn-linked carbohydrate by
weight (10). Therefore, the circulating form of haPL-II
cannot be an aggregate of hormone monomers as has
often been assumed to be the case for high Mr forms
of proteins in the GH-PRL-PL family.
The ability to form high Mr disulfide-bonded complexes is not unique to haPL-II. Human PL (hPL) and
mPL-II form similar complexes when incubated with
human and mouse serum, respectively (10). The major
difference observed for the three PLs is that the conversion to disulfide-bonded complexes in vitro occurs
to a significantly greater extent for haPL-II than for hPL
and mPL-II. Although these experiments have not been
performed with rPL-II, this protein probably behaves
similarly to mPL-II, since no significant amount of high
Mr forms are present in the placenta or in the blood (9).
With regard to the enhanced ability of haPL-II to form
intermolecular disulfide bonds, the most obvious structural difference between haPL-II and the other PLs is
the presence of an additional pair of Cys residues.
The four Cys residues of haPL-II which correspond
to those of mPL-II and rPL-II (at positions 51,166,183,
and 191) are conserved in all known members of the
GH-PRL-PL family (12). In addition, haPL-II contains a
pair of Cys residues not present in the other PL-Ms.
These occur at residue 21 (Asn in mPL-II and Tyr in
rPL-II) and residue 42 (Trp in mPL-II and rPL-II). Like
haPL-II, mammalian PRLs have an additional pair of
Cys residues. The positions of these Cys residues in
PRL are highly conserved (12), with both occurring
within the first 11 amino acid residues of the mature
protein. Currently, complete amino acid sequences are
available for 18 GHs and 13 PRLs (12) and eight placental proteins (excluding haPL-II) related to them (11).
None of these proteins contain Cys residues corresponding to those at positions 21 and 42 of haPL-II.
The four Cys residues of hPL form two intramolecular
disulfide bonds in the monomeric hormone, Cys 53-
Hamster PL-II cDNA
Cys165 and Cys 182-Cys189 (13). Presumably, analogous disulfide bonds (Cys 51-Cys 166 and Cys 183Cys191) are present in mPL-ll and rPL-ll. Purified, monomeric haPL-ll does not contain any sulfhydryl groups
(10) and therefore contains three disulfide bonds in an
unknown arrangement. When purified haPL-ll is incubated with serum in vitro, one or more of these disulfides is disrupted to form an intermolecular disulfide
bond with a serum protein, generating the high Mr
disulfide-bonded form of haPL-ll. It is not possible at
this time to predict which of the Cys residues of haPLII participate in the formation of an intermolecular disulfide bond.
It has become increasingly clear that the placenta is
the source of a number of proteins which, although
obviously structurally related to pituitary GH and PRL,
have subtle or not so subtle structural variations compared to GH and PRL (11). The finding that haPL-ll
contains a uniquely positioned pair of Cys residues
introduces yet another type of variation. As for the
other structural variations, the ultimate functional consequences of this alteration in structure are unknown.
There is in this case, however, a strong correlation
between a relatively small change in structure and a
gross alteration in the mechanism by which the hormone acts. Thus, while haPL-ll and mPL-ll have a 70%
amino acid sequence identity, haPL-ll circulates primarily as a complex with a serum glycoprotein and mPL-ll
circulates primarily as a monomer. The exact nature of
the circulating haPL-ll complex, the mechanism by
which it is formed, and the effect of this process on the
functional properties of PL-II in the hamster remain to
be elucidated.
Acknowledgments
We thank Linda Ogren and Gudmundur Thordarson for their
comments on the manuscript.
Received June 28,1989. Revision received August 3,1989.
Accepted August 4,1989.
Address requests for reprints to: Dr. Frank Talamantes,
Thimann Laboratories, University of California, Santa Cruz,
California 95064.
1713
This work was supported by NIH Grants HD-14966 and
MBRS-RR08132 and NSF Grant DCB-8602865.
* Recipient of a MARC undergraduate fellowship.
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