Download Full-Length Complementary DNA and the Derived Amino Acid

BIOLOGY OF REPRODUCTION 66, 1723–1728 (2002)
Full-Length Complementary DNA and the Derived Amino Acid Sequence
of Horse Uteroglobin1
Frank Müller-Schöttle,3 Agata Bogusz,3 Joachim Grötzinger,4 Andreas Herrler,3 Claudia A. Krusche,3
Karin Beier-Hellwig,3 and Henning M. Beier2,3
Department of Anatomy and Reproductive Biology,3 and Department of Biochemistry,4 Medical School,
RWTH University of Aachen, D-52057 Aachen, Germany
ABSTRACT
After its original description as a steroid-dependent protein
in the rabbit uterus, uteroglobin became one of the best characterized proteins. However, detailed knowledge of its physiological role remains an enigma. In this study we investigate how
its structure is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis
showed that in horses, the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis
demonstrated that the dimeric form of uteroglobin is found predominantly in biological compartments. Using a RACE-PCR
technique, we cloned and sequenced the full-length cDNA (473
base pairs) that encodes equine uteroglobin. The nucleotide sequence was shown to characterize the primary structure of this
protein. This enabled us to add equine uteroglobin to a comparative amino acid alignment of 8 other uteroglobin molecules,
and finally, to unravel 14 evolutionary completely conserved
amino acids. We summarize these results with a computer-based
3-D model of horse uteroglobin, and discuss new concepts on
the physiological role of uteroglobin, in particular as a specific
binding protein.
female reproductive tract, implantation, male reproductive tract,
progesterone, prostate, uterus
INTRODUCTION
Uteroglobin (UGB) was first described by Beier [1, 2]
as a steroid-dependent protein in uterine secretions during
the preimplantation phase and as a major component of
blastocyst fluid in the rabbit. Independently, Krishnan and
Daniel [3] described this protein in rabbit uterine fluid and,
after investigating it in in vitro culture of early rabbit embryos, coined the name blastokinin. From the beginning it
was identified as an unusually small, globular endometrial
secretory protein, and is now defined as a member of the
secretoglobin gene family [4]. UGB proved to be regulated
by progesterone [2, 5, 6] and to be a progesterone binding
protein [7, 8]. The physico-chemical properties of UGB
have been well-characterized, including its crystal structure
and molecular biology (for reviews see [9–11]). The gene
encoding for UGB was first described in rabbits [12] folThis work was supported by START: Forschungsschwerpunkt ‘‘Molekulare
Endokrinologie’’ (TP 6/2000) from the University of Aachen Medical
School, with additional financial support from Schering Aktiengesellschaft, Berlin (Gender Health Care Research).
2
Correspondence: Henning M. Beier, Department of Anatomy and Reproductive Biology, Medical School RWTH Aachen, Wendlingweg 2, D52074 Aachen, Germany. FAX: 49 241-8082508;
e-mail: [email protected]
1
Received: 24 September 2001.
First decision: 17 October 2001.
Accepted: 8 January 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
lowed by equivalent findings in hares [13], humans [14],
rats [15], mice [16], hamsters [17] and pigs [18]. Numerous
investigations of its distribution and its hormone-regulated
expression have been conducted, but the physiological role
of UGB remains an enigma. Evolutionary data exist for
macaques [19]; but similar data do not exist for horses.
More recently, horse UGB was discovered and described
as a major progesterone-dependent secretory protein of the
endometrium of ovarectomized mares [20].
After its original description in the rabbit uterus, UGB
was detected in other organs, particularly in epithelia of the
respiratory, gastrointestinal, and genitourinary tissues of
both genders. In rabbits [21] and later in humans [22], UGB
equivalent molecules were described in the pulmonary epithelium and secretion of the lung, where the origin of UGB
could be attributed to Clara cells. In order to determine and
understand the function of UGB, we considered its structure, how its quantitative appearance varies in organs, that
it is conserved in mammalian species, and how the tissuespecific expression of the molecule could be traced. With
this aim in mind, we cloned and sequenced the full-length
cDNA to encode equine UGB. The nucleotide sequence
enabled us to deduce and characterize the primary structure
of the protein and, on the basis of crystallographic data
from rabbit and human UGB molecules [23, 24], we created
a computer-based three-dimensional model of horse UGB.
MATERIALS AND METHODS
Sample Collection
Tissues and organs (liver, kidney, heart, thyroid gland, adrenal gland,
spleen, pituitary gland, lung, uterus, and prostate) from adult horses were
obtained from a local slaughterhouse immediately after the animals were
killed. Tissues were cut into small pieces, frozen in liquid N2, and stored
until RNA preparation at 2708C. Lung lavage was obtained by flushing
the entire organ with 0.9% sodium chloride solution. Samples were frozen
in 1-ml aliquots at 2708C until they were analyzed.
Western Blot Analysis
For Western blot analysis we used endometrial biopsies and lung lavage. The protein content of all samples was determined as described by
Lowry et al. ([25]; modified with a DC Protein Assay from Bio-Rad,
Munich, Germany). The samples were subjected to SDS-PAGE using a
5% stacking gel and a 15% resolving gel under nonreducing conditions.
The endometrial biopsies were homogenized and diluted in nonreducing
(without mercaptoethanol) or reducing loading buffer (5% [v/v] mercaptoethanol, 30 mM Tris pH 6.8, 1.5 M urea, 7.5% [v/v] glycerol, 0.5% [w/
v] SDS, and 0.05% [w/v] bromophenol blue). Lung lavage samples of 70
mg protein were freeze-dried and resolved in loading buffer. The samples
were heated at 1008C for 5 min, microcentrifuged, and 30 mg of total
protein in 20 ml of loading buffer was loaded onto the gel. After electrophoresis, the gel was blotted onto a polyvinylidene difluoride membrane
(Sartorius, Epsom, U.K.) by semidry blotting according to Kyhse-Andersen [26] for 45 min at 900 mA. Following blotting, the membrane was
blocked with 5% (w/v) milk powder in PBS-Tween-20 (0.1% w/v) overnight at 48C. The primary antibody (rabbit anti-human uteroglobin, a donation from Dr. Klug, Marburg, Germany) was diluted 1:1000 in 1% milk
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MÜLLER-SCHÖTTLE ET AL.
FIG. 2. Western blot analysis of UGB. Employing a rabbit antiserum
against human UGB, horse UGB can be detected as a specific band.
Under nonreducing conditions UGB can be detected as a 16-kDa dimer.
The 8-kDa monomer is detectable by Western blotting under reducing
conditions.
FIG. 1. Northern blot analysis of UGB in several horse tissues. Expression of UGB is highest in lung and uterus and weak in prostate. Kidney
and liver represent tissues that do not express UGB.
powder in PBS and incubated with the blot at room temperature for 1 h.
After three washes in PBS/Tween, the second antibody (donkey anti-rabbit
labeled with horseradish peroxidase diluted 1:5000 in PBS/Tween; DAKO,
Glostrup, Denmark) was added and left at room temperature for 1 h. After
further rigorous washing, detection of the second antibody was performed
using enhanced chemiluminescence reagents (Amersham, Little Chalfont,
U.K.; horseradish peroxidase induced the chemiluminescent reaction) and
exposure to x-ray film (X-Omat AR; IBI-Kodak Ltd, Cambridge, U.K.)
for 1–3 min.
Preparation and Analysis of RNA
band of approximately 400 bp was cut out and the DNA recovered. The
DNA was ligated to the plasmid pGEM-T easy (Promega) and propagated
into E. coli DH5a. Plasmid DNA was prepared and the DNA insert was
sequenced (SeqLab, Göttingen, Germany). From the obtained sequence
we made use of the last 21 nucleotides for a specific reverse primer that
we used in the 59 RACE-PCR protocol according to the manufacturer’s
instructions. The PCR product was subjected to electrophoresis and the
same procedure was created as described before, with the 39 RACE-PCR
product (cloning and sequencing). The first amino acids of mature equine
UGB were verified by Edman degradation (Knauer protein sequencer).
Sequence Data Analysis
The cDNA and the deduced amino acid sequence of horse UGB was
compared with those of other species using the ClustalW computer program. The phylogenetic tree was created with the JalView computer program. The computer design of the tertiary structure of UGB protein was
composed using the coordinates published by Umland et al. [24] on the
human UGB molecule.
Total RNA was extracted using RNAzol (WAK Chemie, Bad Soden,
Germany) according to the method of Chomczynski and Sacchi [27], and
according to the manufacturer’s directions. RNA concentrations were determined spectrophotometrically by absorption at 260 nm. For Northern
blot analysis, RNA (4–8 mg) was separated by electrophoresis using a
1.2% agarose gel containing formaldehyde and ethidium bromide. After
electrophoresis, gels were photographed under UV light to estimate the
amount of ribosomal 28S and 18S, and the integrity of the RNA. The
RNA was transferred by capillary blot to nylon membranes and immobilized by UV radiation. Blots were hybridized using a horse cDNA probe
(400 base pairs [bp], a result of the rapid amplification of cDNA ends and
polymerase chain reaction; RACE-PCR). The probe was random primed
with digoxy dUTP with the digoxigenin DNA labeling kit (Roche, Germany) according to the manufacturer’s directions. Hybridization with the
random primed labeled probe (10 ng probe/ml hybridization solution) was
carried out at 428C overnight. After stringently washing the blots (0.53
SSC and 0.1% SDS), the specific hybridized probe was detected by an
antidigoxigenin antibody conjugated with alkaline phosphatase (Roche,
Germany) and the chemiluminescence substrate CSPD (Roche, Germany).
Earlier studies on UGB in the horse supported the present research goal of analyzing the tissue-specific expression of UGB. Northern blot analysis of RNAs isolated from
horse liver, kidney, heart, thyroid gland, adrenal gland,
spleen, pituitary gland, lung, uterus, and prostate indicated
that its highest expression was in lung tissue, followed by
uterus, with weak expression in the prostate. UGB mRNA
was not detected in other organs. Figure 1 demonstrates the
remarkable expression in lung and uterus, whereas kidney
and liver did not express detectable levels of UGB mRNA.
Cloning of cDNA
Western Blot Analysis
Reverse transcription and cDNA synthesis of total RNA from horse
lung and endometrial samples was performed using the Biometra Personal
Cycler (Göttingen, Germany). All additives (without the oligo(dT) primer)
used for the mixture for reverse transcription (20 ml/tube) were from
Roche (Germany). The RNA (1 mg/tube) was heated for 10 min at 658C
and then kept at 48C for 5 min. After adding the RNA to the reaction
mixture (4 ml 53 DNA reaction buffer, 1 mM dNTP mixture, 1.6 mg
oligo(dT) primer, 20 units RNase inhibitor, and 20 units of reverse transcriptase) the samples were reverse-transcribed at 378C for 1 h.
The specific cDNA for horse UGB was cloned using the FirstChoice
RLM-RACE kit (Ambion) for 59 RACE-PCR and NotI(dT)18 (Pharmacia)
for 39 RACE-PCR. The 39 end of UGB was amplified first by using
NotI(dT)18 oligonucleotides and the UGB wooble primer, UG1FW (59ACCATGAAG(AC)TCGCCATCAC-39). UG1FW resulted from the mostly conserved nucleotide sequences that resulted from an alignment with
the already described UGB cDNA from other species. The reaction was
carried out in a final volume of 50 ml of the PCR reaction buffer (1.5 mM
MgCl2, 0.2 mM dNTP mixture, 0.2 pmol UG1FW primer, 2 pmol NotId(T)18 primer, 0.5 units Taq polymerase, and 2 ml of cDNA). Thermocycling was performed in 35 cycles, with 30 sec at 948C, 45 sec at 508C,
60 sec at 728C, and a final extension of 3 min at 728C. Ten microliters of
each PCR product was subjected to electrophoresis on a 1.2% agarose gel
and stained with ethidium bromide. After electrophoresis the gel was
placed on a UV transilluminator and photographed. The amplified DNA
Western blot analysis (Fig. 2) indicates that horse UGB
is a dimer (16 kDa) just as in other mammalian species.
The monomer form (8 kDa) was detected in homogenized
endometrial biopsies diluted in a reducing loading buffer
(with mercaptoethanol) before SDS-PAGE electrophoresis.
To conserve the natural dimer form of UGB, the same homogenate was diluted in a nonreducing loading buffer
(without mercaptoethanol) before SDS-PAGE electrophoresis.
RESULTS
Tissue-Specific Expression of Equine UGB
Full-Length cDNA Cloning of Equine UGB
One goal of this investigation was to use equine UGB
to construct a computer-based alignment with the nucleotide sequences of the UGB cDNAs from other species. The
cDNA alignment showed that the ATG start codon and a
few following nucleotides are very well conserved among
species. A synthetic nucleotide with this conserved sequence (UG1FW) and an oligo(dT) primer was used in a
39 RACE-PCR with cDNA from equine lung and uterus.
The product resulted in a band of approximately 400 bp.
HORSE UTEROGLOBIN CDNA AND AMINO ACID SEQUENCE
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FIG. 3. The full-length cDNA is described by the three-letter codes for the
amino acid sequence noted underneath by
the single letter code. ATG (translation
start signal) and AATAAA (polyadenylation
signal) are boxed. The arrow shows the
cutting site for the signal peptide sequence. The cysteins at positions 3 and 69
from the mature protein are marked.
Cloning and sequencing of this product indicated that an
almost full-length cDNA for UGB had been isolated. To
obtain the 59 end of the cDNA we used a specific reverse
primer from the 39 end in a 59 RACE-PCR protocol. The
product resulted in a band of approximately 500 bp. Cloning and sequencing of this product indicated that the fulllength cDNA (submitted to GenBank, accession number
AF372660) for equine UGB had been isolated (Fig. 3).
Amino Acid Sequence of Equine UGB
The cloned cDNA for equine UGB codes for a protein
of 91 amino acids. The first 21 amino acids correspond to
the signal peptide and the last 70 amino acids to the mature
secretory peptide. The site of rupture of the signal peptide
was deduced by comparing it with the typical features of
UGB from other species and by amino acid sequencing the
first four amino acids of the mature equine protein (Fig. 4).
Because the first four sequenced amino acids (glycine, isoleucine, cysteine, and glutamic acid) are identical to the
deduced first amino acids of the mature protein as coded
from the cDNA, we confirmed the cutting site of the signal
peptide and, at the same time, the correctness of our deduced amino acid sequence derived from the cloned fulllength cDNA.
Amino Acid Sequence Homology Study
The amino acid sequence of horse UGB shows homology to UGB from pigs (60%), monkeys (57%), humans
(53%), rabbits (51%), hares (49%), rats (45%), mice (41%),
and hamsters (41%). The amino acid alignment of the protein (Fig. 4) and the phylogenetic tree (Fig. 5) revealed that
the structure of horse UGB is most similar to the families
of Artiodactyla (pigs), Primates (humans and macaques)
and Lagomorpha (hares and rabbits). But it significantly
differs from that of Rodentia (mice, rats, hamsters), in
which the signal peptide is shorter and the mature peptide
consists of 77 amino acids compared to that of horses and
other species, for which the mature protein consists of 70
amino acids.
With the known peptide sequences of mature UGB we
created a phylogenetic tree (Fig. 5) for UGB with the
JalView computer program. The Rodentia, Primates, and
Lagomorpha were grouped together. Artiodactyla (pigs) and
Perissodactyla (horses) could not be grouped together because for each, only one UGB peptide sequence has been
described. As shown in Figure 5, primates are more closely
related to pigs and horses than to mice or rats.
Completely Conserved Amino Acids Among Species
The alignment of the amino acid sequences indicated
that some amino acids are completely conserved in UGB
in all species. These completely conserved amino acids
were in the following positions in horse UGB: cysteine at
3 and 69; phenylalanine at 6; leucine at 13, 48, 59, and 68;
proline at 30; glutamine at 40; lysine at 42 and 58; aspartic
acid at 46; isoleucine at 63; and serine at 66. The computer
design of the tertiary structure of UGB protein was determined using the crystallographic data of Morize et al. [23]
and Umland et al. [24], to demonstrate the equally globular
character of horse UGB (Fig. 6). Every completely conserved amino acid (not considering cysteines at positions 3
and 69, which are required for the disulfide bridges to form
FIG. 4. Amino acid alignment of horse UGB with 8 other mammalian UGBs. The amino acid sequence of horse UGB was compared with other
sequences for UGB in different species using the ClustalW computer program. *, Amino acids that show 100% homology. The signal peptide for
macaque UGB has not been reported.
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MÜLLER-SCHÖTTLE ET AL.
FIG. 5. Phylogenetic tree of UGB. The tree was composed using the
JalView computer program, which is based on the unweighted pairedgroup method analyze (UPGMA) method.
a dimer) are allocated at the centrally formed pouch. It is
interesting that other investigators found that this ‘‘pouch’’
is able to bind progesterone or similar lipophilic molecules
[24]. Figure 4 shows these 14 amino acids located mainly
between positions 40 and 68. The characteristic cysteine
residues at positions 3 and 69 are essential for connecting
the two monomeric chains to form the antiparallel chains
in dimeric UGB.
DISCUSSION
More than 30 yr after the discovery of UGB as an endometrial secretory protein, several questions about UGB
are still unanswered. To gain more information about this
protein we performed a study of the structure and tissuespecific expression in the horse because some years ago,
we had described the production of UGB in equine endometrium and its regulation by progesterone [20]. This present study, to the best of our knowledge, revealed for the
first time the full-length cDNA sequence, the deduced complete amino acid sequence, a computer-designed tertiary
structure, and the tissue-specific expression of equine UGB.
Expression and regulation of UGB in equine endometrium shows remarkable similarities to its well-known production and secretion by rabbit endometrium. No other
mammalian species (other than rabbits) are known to express UGB in comparable abundant amounts during progesterone dominance during the ovarian cycle and during
early pregnancy [20]. Further striking similarities have been
demonstrated before on the experimental findings after progesterone substitution in ovarectomized females [20]. As in
rabbits, the endometrial production and release of UGB into
the uterine secretion is induced within a few days after
initiation of treatment with progesterone or a synthetic progestagen. This striking similarity in UGB expression in the
horse, compared to the rabbit, led to our detailed studies
on the molecular structure of equine UGB and on its fulllength cDNA.
FIG. 6. Computer-designed tertiary structure of horse UGB. In this dimeric structure, the two monomers are depicted as blue and green ribbons. The 14 completely conserved amino acids are shown in orange
side chains, indicating their allocation to the so-called ‘‘binding pouch’’
of the dimeric UGB molecule. The lower segment of the binding pouch
is characterized by the phenolic side chains of phenylalanine (position 6
of each subunit). The other conserved amino acids are either strong acidic
side chains (aspartic acid at 46, glutamine at 40) or nonpolarized side
chains, such as leucine (positions 13, 48, 59, and 68) and proline (position 30) or isoleucine (position 63). Serine (position 66) contains an alcoholic hydroxyl side chain, which may be important for certain binding
affinities. Lysine (positions 42 and 48) is characterized by its basic amino
side chain, perhaps interacting with the acidic side chains at positions 40
and 46.
One comparison of the equine UGB amino acid sequence was made with the UGB amino acid sequences from
other species. As Figure 4 demonstrates, 14 amino acids of
the mature UGB are completely conserved among all 9
species investigated. The cysteines at positions 3 and 69
are completely conserved, because they combine the 2
UGB monomers by their disulfide bridges. Lysine at position 42 has been shown to be extremely important for structural stability, because in silico mutations resulted in a total
loss of molecular stability [28].
Most of the other evolutionarily conserved amino acids
have been shown to line the inside of a hydrophobic pocket
that forms between the two subunits of the dimeric UGB.
TABLE 1. Comparison of ‘‘topohydrophobic’’ positions according to Callebaut et al. (2000) of strong hydrophobic amino acids in the UGB molecule of the rabbit, human, and horse.
Phenylalanine*
Isoleucine
Leucine*
Leucine
Tyrosine
Leucine
Phenylalanine*
Methionine
Valine
Leucine
Leucine*
Isoleucine
Leucine
Isoleucine*
Leucine*
Rabbit
Human
Horse
F6
I 10
L 13
L 14
Y 21
L 25
F 28
M 41 (mutation)
V 44 (mutation)
L 45 (mutation)
L 48
I 56
L 59
I 63
L 68
F6
I 10
L 13
L 14
Y 21
L 25
F 28
L 41
L 44
V 45
L 48
I 56
L 59
I 63
L 68
F6
I 10
L 13
F 14 (mutation)
F 21 (mutation)
V 25 (mutation)
F 28
L 41
L 44
V 45
L 48
I 56
L 59
I 63
L 68
* Completely conserved amino acids among all UGB sequences reported.
HORSE UTEROGLOBIN CDNA AND AMINO ACID SEQUENCE
This hydrophobic pocket can bind several lipophilic molecules [24, 29–31]. The results described here support the
hypothesis that UGB serves as a carrier protein, because
85.7% of the conserved amino acids over nine species are
located within the hydrophobic pocket. Actually, the first
described ligand was progesterone [32], but the biological
significance of this binding is not known. After the first
description and isolation in rabbit uterus [1–3] it was speculated that UGB might be a carrier or a scavenger of steroids [11]. In vitro experiments demonstrated that threonine
60 and tyrosine 21 are important for binding progesterone
to UGB in rabbits, mice, and rats [29, 30]. In humans, pigs,
and horses, UGB has no threonine at position 60. In human
UGB, a limited binding capacity of progesterone could be
shown in vitro, because the tyrosine at position 21 remains
[30]. In amino acid sequence of equine UGB, the two amino acids are mutated from tyrosine at position 21 to phenylalanine, and from threonine at position 60 to methionine.
This leads us to speculate that equine UGB may not bind
progesterone, but may bind other lipophilic molecules.
As shown in Figure 6, the lower segment of the ‘‘binding
pouch’’ in UGB is characterized by the phenolic side chains
of phenylalanine (position 6 of each subunit). The other
conserved amino acids are either strong acidic side chains
(aspartic acid at position 46, glutamine at position 40) or
nonpolarized side chains, such as leucine (positions 13, 48,
59, and 68) and proline (position 30) or isoleucine (position
63). Serine (position 66) contains an alcoholic hydroxyl
side chain, which may be important for certain binding affinities. Lysine (positions 42 and 48) is characterized by its
basic amino side chain, perhaps interacting with the acidic
side chains at positions 40 and 46. In Table 1 these strong
hydrophobic amino acids are shown lining the binding
pouch of the dimeric UGB, clearly indicating that the biological ligands of UGB are strongly lipophilic, similar to
phospholipids, steroids, and inositols. The six asterisks in
Table 1 indicate six completely conserved amino acids
among all UGB sequences reported.
The most stimulating observations were made by Umland et al. [24], who indicated that the two phospholipid
molecules, phosphatidylinositol and phosphatidylcholine,
appear as ligands in isolated UGB molecules from human
lung lavage. It may be scientifically rewarding to follow
this lead, because the comparison of ‘‘topohydrophobic’’
positions of strong hydrophobic amino acids in the UGB
molecule all line the so-called ‘‘binding pouch’’ of the dimeric UGB [33] (Table 1). It is interesting that UGB binds
lipophilic polychlorinated biphenyls (PCBs). After intraperitoneal injection of PCBs, UGB knockout mice did not
accumulate PCBs in the lung, compared with high accumulations in wild type controls [34], which emphasizes the
function of UGB as a binding and carrier protein. These
results are consistent with other reports that UGB is a secretory protein in Clara cells of all species investigated so
far [15]. It has been speculated that UGB in the lung may
serve as a binding protein for uptake of toxic agents to
prevent further pathogenic actions on the organism.
Tissue-specific expression of UGB varies among species.
In rabbits [1, 2], humans [35–37], and horses [20] UGB
expression has been well-documented in the reproductive
organs, particularly the endometrium, whereas in pigs it
seems to show no or only extremely weak expression in
the uterus [18]. Results of the present study revealed that
horse and pig UGB have very similar amino acid sequences, therefore, tissue specificity is not necessarily correlated
with the assumed amino acid sequence. Furthermore, the
1727
phylogenetic tree of UGB emphasizes that rodents are not
the experimental model of the first choice because their
amino acid sequence homology is dissimilar to that of humans. In contrast, horses, pigs, and rabbits seem to be more
similar to humans and therefore much better experimental
models.
One of the challenging research goals is the investigation
of binding molecules (for example IGFBP 3 or haptoglobin;
[38–40]) that may play a significant role in the events leading to endometrial receptivity and the establishment of
pregnancy, and in their specific action within the so-called
embryo-maternal dialogue. UGB, recognized now as a certain binding or carrier molecule for highly specific lipophilic ligands, could influence endometrial receptivity and
blastocyst implantation.
ACKNOWLEDGMENTS
We thank Sabine Eisner and Bärbel Bonn for their excellent assistance
in molecular biology work and protein analysis, Manfred Dewor for protein sequencing, and Dr. Jörg Klug for providing the UGB antibody and
for his extremely fruitful discussions.
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