From the
Using a monoclonal antibody, IVd4, that recognizes a novel group
of hyaluronan-binding proteins, we have immunoscreened a cDNA library
constructed from embryonic chick heart muscle mRNA. One of the cDNAs
isolated from the library encodes a 29.3-kDa protein homologous to
Cdc37, an essential cell cycle regulatory factor previously
characterized genetically in yeast and Drosophila; this is the
first vertebrate CDC37 gene to be cloned to date. We also
present evidence for the existence of a second chick isoform that is
identical to the 29.3-kDa protein over the first 175 amino acids but is
entirely different at the carboxyl terminus and lacks the IVd4 epitope.
The avian Cdc37 binds hyaluronan, chondroitin sulfate and heparin in vitro, and both isoforms contain glycosaminoglycan-binding
motifs previously described in several hyaluronan-binding proteins.
These findings suggest a role for glycosaminoglycans in cell division
control.
Interactions of the glycosaminoglycan (GAG),
We have attempted to obtain specific
monoclonal antibodies to hyaluronan-binding proteins involved in
embryonic cellular events by screening hybridomas from mice immunized
with hyaluronan-binding protein preparations from chick embryo brain.
Hybridoma clones were selected that produce antibody whose interaction
with antigen is blocked by hyaluronan and hyaluronan oligosaccharides
(Banerjee and Toole, 1991). One of the antibodies obtained, mAb IVd4,
recognizes a novel group of hyaluronan-binding proteins that are
associated with many tissue and cell types, especially in the embryo.
The molecular masses of the major proteins recognized by mAb IVd4 are
One of the tissues enriched in IVd4
antigen is the chick embryo heart. Thus, in the present study we have
used mAb IVd4 to screen a library prepared from chick embryo heart
muscle mRNA and have isolated and characterized a cDNA that encodes one
of the hyaluronan-binding proteins recognized by mAb IVd4.
Interestingly, the protein encoded by this cDNA appears to be a chicken
homologue of Cdc37, a cell cycle control factor previously
characterized in yeast (Ferguson et al., 1986) and Drosophila (Cutforth and Rubin, 1994); this is the first
vertebrate CDC37 gene to be reported. In this publication we
describe its nucleotide and deduced amino acid sequences, its
GAG-binding properties and their potential significance, and provide
evidence for a second splicing isoform that lacks the IVd4 epitope.
Western blots (Towbin et al., 1979) were reacted with mAb IVd4. Hyaluronan binding
was measured by two different methods. In the first method the blot was
reacted with [
Nucleic acid and protein sequence data were analyzed with DNASIS
(Hitachi Co., Tokyo) and University of Wisconsin Genetics Computer
Group software. Sequence comparisons against the Genbank-EMBL and NBRF
data bases were performed using the FASTA program (Pearson and Lipman,
1988). Multiple alignments and statistical analysis of sequence
similarity between two protein sequences was obtained using the Pretty
and the Bestfit programs, respectively, of the University of Wisconsin
Genetics Computer Group (version 7.3).
Aliquots of total
bacterial extract containing the induced fusion protein were separated
by SDS-PAGE and transblotted. The transblots were then overlaid with
mAb IVd4, or with [
The first in-frame termination
codon within pNG13 is located at position 802 and overlaps with the
polyadenylation signal AATAAA found 15 nucleotides before the poly(A)
tail. This cDNA therefore has an unusually short 3`-untranslated region
composed of only 18 nucleotides between the stop codon and the poly(A)
tail. Short 3`-untranslated regions have been noted previously and can
include an overlap between the termination codon and the
polyadenylation signal as found here (Kawajira et al., 1983;
Furukawa et al., 1990; Lustigman et al., 1992). Also,
we are confident that the 3`-sequence obtained is valid since, by
nucleic acid library screening, we have isolated an additional cDNA
whose sequence has an identical 3`-untranslated region to pNG13, and
since reverse transcriptase-PCR using primers spanning the C-terminal
coding and 3`-untranslated regions gives products of the expected size
(data not shown).
Assuming that the open reading frame does initiate
at the codon discussed above, it would encode a 246 amino acid
polypeptide, with a calculated molecular mass of 29.3 kDa. When
expressed in a eukaryotic in vitro translation system, a major
polypeptide of molecular mass,
The 5`-terminal nucleotide residues
of the above cDNAs were found by sequencing to correspond to nt 214 and
442 of pNG13, the two smaller cDNAs being identical. The largest of
these cDNAs, pNG17, lacks 213 nt corresponding to the 5`-end of pNG13.
As stated above, nt residues 214-588 of pNG13 are identical to
the 5`-end of pNG17, but thereafter the sequences are entirely
different. The open reading frame of pNG17 continues for 129 nt and is
then followed by a relatively long, T-rich, 3`-untranslated region of
841 nt. The unique sequence of the pNG17 cDNA is presented in Fig. 4B.
To test whether this polyadenylated cDNA is
truncated at the 5`-end, we employed 5`-RACE, using an antisense primer
corresponding to nt 273-295 of pNG13 and overlapping by 82 bp at
the 5`-end of the 5`-truncated pNG17 clone. This primer directed the
synthesis of a PCR product of 295 bp whose sequence matches exactly
with the 5`-end of pNG13. Assuming, therefore, that the full-length
pNG17 cDNA also originates at the 5`-end of pNG13, the full size of
this cDNA would be 1558 nt, which agrees well with the size of the
major 1700-nt polyadenylated mRNA detected by hybridization with pNG13
in Fig. 3. Indeed, a fragment derived from the unique
3`-untranslated region of pNG17 was found to detect only the 1700-nt
mRNA isoform after rehybridization of the blot in Fig. 3. These
results indicate the existence of two related mRNAs that possess
identical 5` moieties but differ at their 3`-ends, presumably due to
alternative splicing.
Whereas mAb IVd4 recognizes the pNG13-encoded
protein (Fig. 1), the antibody failed to recognize the fusion
protein produced by the pNG17-pBluescript construct after SDS-PAGE and
Western blotting. To further localize the IVd4 epitope in the
pNG13-encoded protein, we have used Western blotting of IPTG-induced
bacterial extracts containing overlapping, carboxyl terminally
truncated mutants of the pNG13 protein. These mutants were produced by
placement of the pNG13 cDNA insert adjacent to the lacZ promoter and in
frame with the m19M13 vector
Despite the great phylogenetic distance
between insect and vertebrate lineages, the Cdc37 sequences of Drosophila and chick show a high degree of conservation at the
amino acid level over the entire length of the shorter avian
polypeptide. Three of the cysteines within the chick sequence (residues
131, 206, and 234) are also present in the same positions in the Drosophila sequence while no cysteines appear in the yeast
counterpart. Another structural similarity between the chick and Drosophila proteins is that both could potentially form
extensive
From the above observations it appears that we have isolated the
chick homologue of the cell cycle protein, Cdc37. However, the fact
that the chick polypeptide is significantly shorter than the Drosophila or yeast forms would suggest that the cDNA isolated
here may represent a different splicing variant than those described in Drosophila and yeast, or that the cloned chick cDNA is not
full-length. The latter seems unlikely because of the reasons discussed
above, i.e. size correspondence with the 900-nt mRNA, lack of
extension of the sequence by 5`- and 3`-RACE, identification of
multiple phage isolates with the same sequence as that presented for
pNG13 in Fig. 4A, and our documentation that this cDNA
contains all the appropriate elements for expression of a eukaryotic
protein. In addition, hypothetical translation of the 63 nucleotides
preceding the initiator codon indicated in Fig. 4A did
not reveal any amino acid sequence homology with the amino terminus of
either the Drosophila or yeast polypeptides, which are
themselves dissimilar to each other in this region. However, the
possibility that the chick mRNA could also initiate further upstream
can still not be conclusively ruled out, especially since no in-frame
termination codon was detected in the 63-nt putative 5`-untranslated
region.
With respect to the possibility of multiple isoforms, first
we have found by Southern blotting of genomic DNA that there is most
likely a single gene expressing Cdc37. Second, we have characterized
another chick isoform, pNG17, corresponding to the abundant 1.7-kb
mRNA. This isoform shares exactly the same 5`-coding region with pNG13,
but its 3`-coding and untranslated regions are entirely different from
pNG13. Interestingly, the unique 3`-coding and untranslated regions of
this isoform also have no significant similarity to any part of the Drosophila or yeast Cdc37 cDNAs. We believe that the pNG13 and
pNG17 cDNAs correspond to mRNAs produced by alternative splicing in the
avian cells. Thus we conclude that the protein encoded by pNG13 is
homologous with the central coding domain of Drosophila and
yeast Cdc37, but that pNG13 encodes a shorter protein than the Drosophila and yeast counterparts. It is not yet fully clear
whether chick cells express a homologous protein similar in size to
these other species in addition to the proteins encoded by pNG13 and
pNG17, whether pNG13 is a truncated cDNA despite our evidence to the
contrary, or whether the chick produces the pNG13 and pNG17-encoded
proteins rather than the isoform found in Drosophila and
yeast. However, since the complete genomic sequence of Drosophila
CDC37 has been reported (Cutforth and Rubin, 1994), we have
compared the exonic boundaries of the Drosophila gene with the
sequence of pNG13. In doing so, we have observed that the 5`-end of the
pNG13 cDNA corresponds exactly with the 5` border of exon 3 of the Drosophila gene. Thus, transcription of chick Cdc37 mRNA may
originate at this position, giving rise to a mRNA that is two exons
shorter than the Drosophila mRNA. Although a detailed analysis
of the chick CDC37 structural gene would be necessary to
confirm this unequivocally, the existence of a second chick Cdc37
isoform, pNG17, whose 5`-end apparently coincides with that of pNG13
when extended to its full-length, supports this hypothesis. The total
length of the extended pNG17 cDNA (1558 bp) agrees closely with the
size of the predominant
The above conclusion that
pNG13 encodes a chick Cdc37 isoform, together with the lack of a
definitive signal sequence at the beginning of the open reading frame,
implies that the pNG13-encoded protein is intracellular. As noted
above, intracellular immunoreactivity with mAb IVd4 has been noted
previously (Banerjee and Toole, 1992), but recognition of this
intracellular protein is presumably not responsible for the inhibitory
action of mAb IVd4 on cell behavior in culture (Banerjee and Toole,
1992; Yu et al., 1992; Toole et al., 1993). As also
noted above, mAb IVd4 recognizes several proteins, the major species
having molecular weights between 35 and 90 kDa. The results presented
here are consistent with the smallest of these proteins being Cdc37.
A significant point of interest is the variable specificity of the
known GAG-binding proteins despite the presence of similar binding
motifs. Link protein and the hyaluronan-binding regions of
proteoglycans are specific for hyaluronan (Hascall, 1977; Yamagata et al., 1986; Goetinck et al., 1987; Perides et
al., 1992; LeBaron et al., 1992). CD44 recognizes only
hyaluronan or chondroitin when present within the cell membrane but
also recognizes chondroitin sulfate when in solution or in artificial
membranes (Underhill et al., 1983; Chi-Rosso and Toole, 1987).
RHAMM recognizes hyaluronan and heparin only (Yang et al.,
1994b), and the protein described here recognizes hyaluronan,
chondroitin sulfate, and heparin. Despite this variability, the
hyaluronan-binding motifs in RHAMM, CD44, and link protein all conform
to the (-B(X
A great deal of evidence has been published
indicating that GAGs are present, at least transiently, in the
cytoplasm and in the nucleus. The types of GAGs shown to be present in
these cellular compartments include hyaluronan, chondroitin sulfate,
dermatan sulfate, and heparan sulfate (Furukawa and Terayama, 1977;
Fedarko and Conrad, 1986; Ishihara et al., 1986; Ripellino et al., 1988, 1989; Hiscock et al., 1994). Of
particular interest is the observation that targeting of a specific
subpopulation of heparan sulfate to the nucleus of rat hepatoma cells
increases markedly under conditions of reduced growth rate and
decreases on stimulation of cell division (Ishihara and Conrad, 1989;
Fedarko et al., 1989). It has also been shown that heparin and
related polysaccharides inhibit the action of Fos and Jun on
transcription events involved in cell cycle progression, and evidence
has been presented suggesting that endogenous nuclear heparan sulfate
may exhibit this regulatory role in vivo (Busch et
al., 1992). Heparan sulfate-heparin is targeted to the nucleus and
elicits similar effects to the above even when added extracellularly
(Fedarko et al., 1989; Pukac et al., 1990). It seems
likely then that GAGs in the cytoplasm or nucleus are involved in cell
cycle regulation and possibly other intracellular events. Binding of
GAG to Cdc37 may mediate one or more of these events.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Al Sabbaj for technical assistance and Dr.
Marion Gordon for helpful comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)hyaluronan, with binding proteins produced by several
types of cells influence their behavior in a variety of ways (Toole,
1991; Turley, 1992; Underhill, 1992; Laurent and Fraser, 1992; Knudson
and Knudson, 1993; Lesley et al., 1993; Sherman et
al., 1994). In particular, cell proliferation is significantly
affected by hyaluronan and other GAGs (Brecht et al., 1986;
Yoneda et al., 1988; Fedarko et al., 1989; Pukac et al., 1990; Rapraeger et al., 1991; Busch et
al., 1992), but the precise mechanisms by which this occurs are
not fully understood.
35, 50, 70, and 90 kDa; the relative amounts of these forms varies
from tissue to tissue (Toole, 1991; Banerjee and Toole, 1991,
1992)
(
)This antibody influences morphogenesis in
various ways (Toole, 1991; Banerjee and Toole, 1992; Yu et
al., 1992; Toole et al., 1993). We have also observed
that high levels of IVd4-reactive antigen sometimes occur
intracellularly as well as extracellularly (Banerjee and Toole, 1992)
but, until the current study was undertaken, the significance of this
finding had not been apparent.
Materials
The
[H]hyaluronan, mAb IVd4, hyaluronan
hexaccharides, and biotinylated hyaluronan were produced as described
elsewhere (Underhill and Toole, 1979; Banerjee and Toole, 1991; Pouyani
and Prestwich, 1994). Hyaluronan was a gift from Anika Research
(Woburn, MA), and chondroitin sulfate and heparin were purchased from
Sigma. Restriction and modification enzymes and the kits used for 5`-
and 3`-RACE experiments were purchased from Life Technologies, Inc.
Kits for immunoscreening and generation of probes by random priming
were from Stratagene (La Jolla, CA). The in vitro transcription and translation kit was from Promega (Madison, WI)
and radionucleotides from NEN (Boston, MA) or ICN Radiochemicals
(Irvine, CA). Nitrocellulose membranes used for plaque lifts and
agarose blotting were from Millipore (Bedford, MA) and Schleicher and
Schuell, respectively. Taq polymerase and the reverse
transcriptase-PCR kit were supplied by Perkin Elmer. Ingredients for
media preparation were from Life Technologies, Inc. Vectastain ABC kits
were from Vector Laboratories (Burlingame, CA). Autoradiography was
done using Kodak XAR x-ray film. Oligonucleotide primers were prepared
in facilities at Tufts Medical School.
Expression Library Screening with mAb IVd4
A chick
embryo cardiocyte library (cDNA made from poly(A) mRNA
ligated into the EcoRI and XhoI sites of UNIZAP XR
and supplied by Drs. R. Markwald and E. Krug, University of S. Carolina
Medical School) was used as a source of cDNA clones. A total of 2
10
plaques were screened with mAb IVd4. Bound
antibody was detected with goat anti-mouse alkaline
phosphatase-conjugated antibody and nitro blue tetrazolium according to
the manufacturer's instructions (Stratagene). Phage clones were
plaque purified after two more rounds of screening; plasmid that was in vivo excised from the phage was then induced for
-galactosidase fusion protein production in liquid culture. Using
the XbaI and KpnI sites present in the polylinker of
the pBluescript KS(+) vector, cDNA inserts were subcloned into
M13mp19 for deletion construction and sequencing.
Nucleic Acid Hybridizations
Nitrocellulose
membranes were prehybridized for 2-4 h in a solution containing
50% deionized formamide, 5 SSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0), 50 mM sodium phosphate (pH
6.5), 5
Denhardt's, 200 µg/ml sheared salmon sperm
DNA, and 0.1% SDS prior to incubation with random-primed probes
(Feinberg and Vogelstein, 1984) at 42 °C overnight in fresh
hybridization solution. The membranes were then washed twice for 30 min
each at 56 °C in 0.1
SSC, 0.1% SDS, and exposed to x-ray
film overnight with an intensifying screen at -80 °C.
Northern Blotting
Total RNA was isolated from
chick embryo heart by the acid guanidinium
thiocyanate-phenol-chloroform extraction method (Chomczynski and
Sacchi, 1987), fractionated in 1.2% denaturing agarose gels containing
2.2 M formaldehyde, and blotted onto nitrocellulose (Sambrook et al., 1989). In order to detect the low abundance 0.9-kb
transcript, it was necessary to hybridize blots containing 20-25
µg of RNA in the presence of 1% dextran sulfate with high specific
activity, antisense probes. To prepare the probe used, single-stranded
DNA (0.5 µg) from a M13mp19 construct containing the entire pNG13
insert was annealed to M13 universal primer in 10 mM Tris-HCl,
10 mM MgCl, 75 mM dithiothreitol, pH 7.5,
at 65 °C for 5 min. The antisense (non-coding) strand of the insert
was homogeneously radiolabeled using DNA polymerase I (Klenow fragment)
and 100 µCi of [
-
P]dCTP plus dATP,
dGTP, and dTTP (0.5 mM each) at 37 °C for 20 min. The
reaction product was subsequently digested with EcoRI which
cuts at the multiple cloning site of the vector. The resulting 0.8-kb
single-stranded fragment was purified by electroelution on a 6%
sequencing gel and used as a probe at 10
cpm/ml of
hybridization solution.
RACE
Poly(A) RNA was obtained by
oligo(dT) chromatography from 15 day chick embryo heart total RNA. 3`-
and 5`-RACE (Frohman et al., 1988) were performed using
reagent kits and modified instructions supplied by the manufacturer
(Life Technologies, Inc.). For 3`-RACE, cDNA synthesis was primed from
25 ng of cardiac mRNA with an oligo(dT)-containing adapter primer,
followed by extension with M-MLV reverse transcriptase. After digestion
with RNase H and purification of the cDNAs, amplification was performed
using a 3`-specific sense primer corresponding to nucleotide positions
685-716 of the pNG13 cDNA presented in Fig. 4and the
universal adapter primer provided by the supplier. A second
amplification was carried out using a nested primer (nt 750-773; Fig. 4) and the universal adapter primer. For 5`-RACE, first
strand cDNA was synthesized from 10 ng of mRNA by incubation with 2.5
pmol of a 5`-specific antisense primer (corresponding to nt
208-234 of pNG13; Fig. 4) and Moloney murine leukemia virus
reverse transcriptase at 42 °C for 30 min. After removal of the
initial primer and mRNA, the cDNA was tailed with dCTP and terminal
transferase. Subsequent AmpliTaq (Perkin Elmer) amplification was done
with nested antisense primers (nt 135-157 and nt 112-137; Fig. 4) and a deoxyinosine-containing anchor primer (Life
Technologies, Inc.).
Figure 4:
Sequences of pNG13 and pNG17 cDNAs and
deduced proteins. The nucleotide and predicted amino acid sequences are
shown. The initiation codon and polyadenylation signals are indicated
by brackets above the nucleotide sequence. GAG-binding motifs
are underlined. A, pNG13. Arrowheads indicate the 5`-end of pNG17 and the point at which its sequence
diverges from pNG13. The epitope for mAb IVd4 lies in the region
denoted by a dotted line below the amino acid sequence. Note
that the polyadenylation signal overlaps the termination codon in
pNG13. B, the unique portion of pNG17 beginning immediately
after nucleotide 588 of pNG13. The numbering of residues begins at
nucleotide 589 since the preceding residues are identical to those
presented in A.
Denaturation, annealing, and extension were
performed for 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72
°C, respectively, for 30 cycles. 3`- and 5`-RACE PCR products were
visualized by ethidium bromide staining and, after subcloning in pCRII
(Invitrogen, Irvine, CA), they were sequenced and compared to the 3`-
and 5`-nt ends, respectively, of the pNG13 cDNA.
Western Blotting and Hyaluronan Binding
Procedures
Expression of cDNA-encoded protein using the
pBluescript vector was induced by growing the bacterial culture to an
OD of 0.5 and then adding 1PTG to a final concentration
of 1 mM. The cultures were grown for an additional 1 h,
pelleted by centrifugation, resuspended in 50 mM Tris-Cl (pH
8.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
10% sucrose, and lysed by sonication. The lysates were mixed with an
equal volume of SDS-PAGE sample buffer and electrophoresis was
performed in 12% SDS-polyacrylamide gels (Laemmli, 1970). The gels were
stained in Coomassie Blue or blotted to polyvinylidene difluoride for
reaction with antibody or with hyaluronan.
H]hyaluronan as described
previously (Banerjee and Toole, 1991). In the second method, the blot
was reacted with biotinylated hyaluronan (Yu and Toole, 1995). The
biotinylated hyaluronan was made using hydrazido-hyaluronan according
to instructions provided by Pierce and also described in Pouyani and
Prestwich(1994). Specificity of interaction in these methods was
confirmed by competition with hyaluronan, hyaluronan oligosaccharides,
and other GAGs.
In Vitro Transcription and Translation
pNG13 in
pBluescript SK vector was used as template with T7 RNA polymerase to
synthesize RNA transcripts in vitro. The RNA was translated in
a rabbit reticulocyte lysate system (Promega, Madison WI) containing
[S]methionine. In vitro translation
products were separated by SDS-PAGE and visualized by fluorography.
Nucleotide Sequencing and Computer
Analyses
Prospective hyaluronan-binding protein phage clones
were in vivo excised to their pBluescript forms and their
inserts were subcloned into M13mp19. Overlapping 3` deletions for each
of the two DNA strands were then generated with T DNA
polymerase by the method of Dale et al.(1985) and sequenced
with Sequenase (U. S. Biochemicals Inc., Cleveland OH). When necessary,
PCR sequencing was performed, and formamide denaturing PAGE was used.
Expression Cloning of cDNA for IVd4
Antigen
Since immunohistochemical staining of chick embryo heart
with mAb IVd4 showed strong immunoreactivity in both the myocardium and
the endocardium, we screened a chick embryo cardiac muscle cell library
for immunoreactivity with mAb IVd4. Of several clones that were
immunopositive over successive rounds of screening, clone -NG13
displayed consistently strong immunoreactivity with IVd4. The phage
clone was isolated and, after in vivo conversion to its
pBluescript plasmid form, it was used for sequencing, mapping, and
preparation of
-galactosidase fusion protein.
H]hyaluronan in the presence
and absence of hyaluronan oligosaccharides (Banerjee and Toole, 1991)
to verify that the protein encoded by the selected cDNA was indeed a
IVd4-reactive, hyaluronan-binding protein. The Western blot with IVd4
revealed three bands of molecular masses 36, 29, and 22 kDa (Fig. 1). The largest of these polypeptides corresponds in size
to that expected for the full-length
-galactosidase fusion
protein. The two smaller polypeptides presumably represent degradation
products of the full-length protein or internally initiated
polypeptides. The [
H]hyaluronan overlay showed a
major peak of [
H]hyaluronan binding that
corresponded exactly in electrophoretic migration to the full-length
IVd4-reactive fusion polypeptide, and a smaller peak of binding that
spread over the region of the two smaller size polypeptides. Since
hyaluronan hexasaccharides competitively inhibit binding of
[
H]hyaluronan to IVd4-reactive proteins (Banerjee
and Toole, 1991), they were included in the reaction mixture as a
control for specificity in the binding assay. The binding of
[
H]hyaluronan to the fusion proteins was
eliminated in the presence of these oligosaccharides (Fig. 1).
Figure 1:
Binding of hyaluronan to pNG13-encoded
protein. Plasmid pNG13 was used to prepare -galactosidase fusion
protein in pBluescript. An aliquot of bacterial lysate obtained after
IPTG induction was electrophoresed, transblotted, overlaid with
10
cpm of [
H]hyaluronan (solid
bars), incubated for 16 h at 4 °C, and washed thoroughly. To
confirm specificity of HA binding, an identical aliquot was
electrophoresed, transblotted, and then incubated with
[
H]hyaluronan in the presence of hyaluronan
hexasaccharides (hatched bars). Each blot was cut into 2-mm
segments for measurement of radioactivity (cpm), which was then plotted
against the distance of migration during electrophoresis. The inset shows a Western blot (W) of a third aliquot after
incubation with mAb IVd4; three reactive bands at
22, 29, and 36
kDa were obtained. When the above experiments were performed on
bacterial extracts without IPTG induction, no
[
H]hyaluronan binding or IVd4 reactivity was
observed (data not shown). The inset also shows the total
IPTG-induced bacterial lysate (L) after electrophoresis and
staining with Coomassie Blue, and a scale to facilitate comparison of
the distance of migration (D) of the IVd4-reactive and
hyaluronan-reactive proteins.
To confirm the hyaluronan-binding data by a direct visual method, we
also used biotinylated hyaluronan to detect binding to discrete protein
bands on the transblots (Yang et al., 1994a; Yu and Toole,
1995). Once more, hyaluronan binding was detected mainly to a
recombinant protein of 36 kDa, and this binding was inhibited by
hyaluronan hexasaccharides (Fig. 2). In this set of experiments,
the ability of other GAGs to compete for hyaluronan binding was also
tested, and it was found that chondroitin sulfate and heparin inhibited
hyaluronan binding to the protein encoded by pNG13 (Fig. 2).
Thus, this protein appears to exhibit general GAG binding properties.
Figure 2:
Specificity of interaction of hyaluronan
with pNG13-encoded protein. Aliquots of bacterial lysate obtained after
transformation with pNG13 were electrophoresed, transblotted, and
incubated for 2 h at room temperature with 10 µg/ml biotinylated
hyaluronan alone (lane 1) or in the presence of 200 µg/ml
unlabeled hyaluronan (lane 2), 200 µg/ml chondroitin
sulfate (lane 3), 200 µg/ml heparin (lane 4), or
1 mg/ml hyaluronan oligosaccharides (lane 5). The arrowhead indicates a 36-kDa hyaluronan-binding protein.
Addition of the various GAGs or HA oligomer inhibited binding of
biotinylated hyaluronan to this protein.
Molecular Characterization of pNG13
The
900-bp insert of clone pNG13, devoid of its poly(A) tail, was
radiolabeled and hybridized with a Northern blot containing chick
embryo heart RNA. Under stringent hybridization conditions, the pNG13
insert recognized two mRNAs: a prominent band of
1700 nt and a
faint band of
900 nt (Fig. 3). Although the signal obtained
at
900 nt was weak, we believe that this rare mRNA corresponds to
the pNG13 cDNA, and that pNG13 is virtually full-length, for the
following reasons. First, the size of the pNG13 cDNA and its encoded
polypeptide are consistent with a 900-nt mRNA. Second, 5`- and 3`-RACE
failed to extend the pNG13 cDNA sequence except for two additional
residues at the 5`-end. Third and most importantly, the cloning and
characterization of a second cDNA, corresponding in size to the
prominent 1700-nt mRNA but having an entirely different 3`-sequence,
indicate that this larger mRNA and the relatively minor 900-nt mRNA may
arise from the same gene by alternative splicing; details of this cDNA
(pNG17) which encodes a protein lacking the mAb IVd4 epitope are given
below.
Figure 3:
Northern blot of chick embryo heart RNA
with pNG13 cDNA. Fifteen day chick embryo heart RNA (25 µg) was
hybridized with a homogeneously labeled single-stranded pNG13 probe. A
major 1.7 kb and a minor
0.9 kb species of RNA hybridize with
pNG13 (arrowheads).
Nested M13 deletions were generated to accurately determine
the full sequence of pNG13 (Fig. 4A). The 894-bp,
polyadenylated cDNA contains a single uninterrupted open reading frame
of 738 bp, beginning at the first ATG codon at nucleotide position 64.
This ATG codon is in frame with the -galactosidase fusion protein
and is surrounded by nucleotides conforming to the Kozak consensus for
eukaryotic translation start signals (Kozak, 1987, 1991). The deduced
amino acid sequence following this potential initiation codon does not
have the properties of a signal peptide that would be expected for a
cell surface or extracellular GAG-binding protein, implying that it is
probably intracellular. Although mAb IVd4 clearly detects extracellular
or cell surface antigen(s) and elicits effects that would depend on
such localization, it also recognizes intracellular antigen(s)
(Banerjee and Toole, 1992). Thus the putative intracellular
localization of the IVd4-reactive protein encoded by pNG13 is
consistent with our past findings but confirms that the IVd4 epitope is
present on more than one protein.
31 kDa, was produced (Fig. 5). The largest of the bacterial fusion proteins obtained
by expression in pBluescript was
36 kDa (Fig. 1), which also
corresponds well to an encoded product of 29.3 kDa after subtraction of
the
-galactosidase fragment of the fusion protein and the
following in-frame sequence corresponding to the 5`-untranslated region
of the cDNA (a total of 7.4 kDa). The expected sizes were also obtained
in other prokaryotic expression systems, including pGEX which has been
used to prepare purified fusion protein. Therefore the open reading
frame presented in Fig. 4A is compatible with the size
of the fusion proteins produced in bacterial systems and with the
protein produced by in vitro translation of pNG13. This
sequence was also found to conform to various established criteria
(test code, codon preference) for avian-specific cDNAs.
Figure 5:
In vitro translation of pNG13.
After transcription and translation, the protein products were
electrophoresed and visualized by fluorography. Lane 1,
antisense template, i.e. transcribed with T RNA
polymerase; lane 2, sense template, i.e. transcribed
with T
RNA polymerase. A major product of
31 kDa was
synthesized from the sense transcript only.
Isolation of a Splicing Isoform Related to
pNG13
To search for additional clones related to pNG13, we used
the entire pNG13 cDNA as a probe in library hybridization experiments.
This approach led to identification of four additional positive cDNA
clones. These cDNA clones, which were all polyadenylated, were
characterized by restriction mapping, Southern hybridization, and
nucleotide sequencing. One of the clones was found to be a 5` truncated
version of the pNG13 cDNA whereas the other three clones possess a 3`
proximal region that is entirely different to pNG13. Further analysis
revealed that these three cDNAs have overlapping, exactly matched
sequences with each other, and they are all identical to pNG13 up to
nucleotide 588. Beyond residue 588, the three clones differ from pNG13
in that they have a unique 3`-sequence exhibiting no homology with the
corresponding region of pNG13.
-galactosidase gene, followed by
exonuclease digestion to create overlapping 3` deletion M13 subclones.
The IVd4 epitope was found to lie within a 15 amino acid region near
the carboxyl terminus of the pNG13-encoded protein (Grammatikakis and
Toole, 1995). The position of this epitope lies in the region unique to
pNG13 (Fig. 4), thus explaining its absence in pNG17 and our
failure to detect this cDNA in our initial immunoscreening.
The pNG13 cDNA Encodes an Avian Homologue of
Cdc37
Computer searches of the available protein sequences
revealed strong identity within the polypeptide sequences encoded by
pNG13, pNG17, and Drosophila Cdc37 (Cutforth and Rubin, 1994),
as well as, albeit to a lesser extent, Cdc37 from yeast (Ferguson et al., 1986). The aligned polypeptides are compared in Fig. 6. Homology with Drosophila and yeast Cdc37 extends
over the major part (amino acids 3-242) of the 246 amino acid
chick pNG13 sequence, after which the polypeptides are dissimilar from
each other. Notably, this is the area of homology between the Drosophila and yeast polypeptides, outside of which they are
also dissimilar. Since pNG17 is identical with pNG13 only up to amino
acid residue 175, this is also the extent of homology between the
pNG17-encoded protein and Cdc37. No significant homologies were found
between the unique, carboxyl-terminal region of pNG17 or its
3`-untranslated region (Fig. 4B) and Cdc37 or other
cDNAs in the data bank. Thus further comparisons are made only between
the pNG13-encoded protein and Cdc37.
Figure 6:
Comparison of yeast and Drosophila Cdc37 with the pNG13-encoded protein. Comparative alignment of the
pNG13-encoded protein (Ccdc37) with the central portions of Drosophila (Dcdc37) and yeast (Ycdc37) Cdc37
polypeptides, as predicted from the cDNA sequence data
(EMBL/GenBank/DDJB accession numbers L32834 and X04288, respectively). Numbers on the right correspond to last amino acid residue of
each sequence compared. Asterisks indicate residues that are
identical in all three polypeptides; capital letters represent
residues that are identical or highly conserved in two of the three
sequences. GAG-binding motifs in the chick protein are indicated by lines above the sequence.
Over the area of homology
between pNG13 and Cdc37 (Fig. 6), the chick polypeptide
(Met to Pro
) is 57% identical to a
corresponding, 245-amino acid, central portion of the 389 amino acid Drosophila sequence (Glu
to
Pro
), and 22% identical to a 295-amino acid region within
the 440 amino acid yeast sequence (Leu
to
Ile
). Considering conserved amino acid substitutions, the
degrees of similarity rise to 62% between chick and Drosophila and 30% between chick and yeast. In comparison, the identity
between Drosophila and yeast sequences is also 22%, and the
degree of similarity is 30%. It appears, therefore, that the yeast
sequence has diverged at similar rates from each of the two metazoan
counterparts. The yeast polypeptide is identical over 15% of its amino
acid residues with the consensus between chick and Drosophila sequences. However, 29% of the residues within the yeast sequence
are identical to one or the other of the chick or Drosophila sequences at any given amino acid position in the aligned
polypeptides. This value increases to 40% by including conservative
amino acid substitutions.
-helical domains throughout their length. An
-helical structure has also been predicted for yeast Cdc37
(Ferguson et al., 1986). In addition, Cutforth and Rubin
(1994) have reported that the Drosophila Cdc37 homologue was
able to complement the corresponding cdc37 mutation in yeast cells.
1700-nt polyadenylated mRNA species
detected by Northern analysis. In contrast, if transcription of the
chick CDC37 gene begins at the same position as in Drosophila, a 2000-2100-nt polyadenylated mRNA would
then be predicted in the chick tissues, well above the size of the
1700-nt mRNA detected. It is also noteworthy that the 5`-ends of the Drosophila and yeast cdc37 cDNAs are dissimilar from each
other both in their sequences and in their lengths (Ferguson et
al., 1986; Cutforth and Rubin, 1994).
Comparison of the Putative Chick Cdc37 with other Known
GAG-binding Proteins
The binding regions of many
hyaluronan-binding proteins lie within characteristic loops that are
often tandemly repeated (Goetinck et al., 1987; Zimmermann and
Ruoslahti, 1989; Rauch et al., 1992; Jaworski et al.,
1994). This type of structure was not detected within chick Cdc37.
However, our analysis did reveal the hyaluronan-binding motif
(-B(X)B-), where B is arginine or lysine, X is any
non-acidic amino acid, and at least one additional basic amino acid
lies within or adjacent to the motif (Yang et al., 1994a). In
chick Cdc37, this motif occurs between amino acid residues Arg
to Arg
. Yang et al. (1994a) also found
that significant hyaluronan binding was obtained to polypeptides where
slight changes were introduced into the motif
(-B(X
)B-), provided basic residues remained within
or flanking the motif. Thus the sequence between residues Lys
and Arg
which has 4 basic residues within a
(-B(X
)B-) motif could most likely also mediate
hyaluronan binding. These two motifs are present in both the pNG13- and
pNG17-encoded proteins. Considerable evidence has been obtained that
motifs of this nature are necessary for hyaluronan binding in several
GAG-binding proteins, whether or not they contain the above mentioned
tandemly repeated loops (Yang et al., 1994a). Heparin-binding
proteins also employ a variety of binding motifs that are highly
enriched in basic amino acids and non-random in sequence (Cardin and
Weintraub, 1989; Sobel et al., 1992; Pratt et al.,
1992). In fact, the two (-B(X
)B-) motifs present
in the RHAMM (receptor for hyaluronan-mediated motility) protein, where
they were first characterized, bind both hyaluronan and heparin but not
chondroitin sulfate or dermatan sulfate (Yang et al., 1994b).
)B-) sequence (Yang et al.,
1994a). It is not yet clear whether separate sequences within the
proteins influence the specificity of binding of this motif, whether
the motif itself varies in its specificity according to its own precise
sequence, or whether factors such as conformation or interactions with
other molecules confer the different specificities. The last
possibility is supported by the influence of carbohydrate side chains
and interactions within the cell membrane on the ability of CD44 to
bind hyaluronan (Lesley et al., 1993; Lokeshwar and
Bourguignon, 1991) and on the specificity of GAG binding to CD44
(Chi-Rosso and Toole, 1987).
Potential Role of a GAG-binding Protein in Cell Cycle
Control
Cdc37 is an essential component of cell cycle regulation
in yeast (Reed, 1980, 1992; Ferguson et al., 1986), and it may
also play a role in certain differentiation events (Simon et
al., 1991; Cutforth and Rubin, 1994). The biochemical function of
Cdc37 is unknown, but genetic evidence strongly suggests that Cdc37
influences the activity of p34 kinase and consequently
cell cycle progression (Reed, 1992; Boschelli, 1993; Cutforth and
Rubin, 1994). In this study we show that a chick homologue of Cdc37
exhibits characteristic properties of a GAG-binding protein. The
putative GAG binding regions of the chick Cdc37 are well conserved in
the Drosophila protein but only partially in yeast (Fig. 6). Thus, considering the phylogenetic distance between
these organisms, it seems reasonable to suppose that binding of GAGs to
Cdc37 may have a significant physiological role, at least in the
metazoan species.
/EMBL Data Bank with accession number(s) U20281 and U25026.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.