©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Glycosaminoglycan-binding Protein Is the Vertebrate Homologue of the Cell Cycle Control Protein, Cdc37 (*)

Nicholas Grammatikakis , Aliki Grammatikakis , Masahiko Yoneda (§) , Qin Yu , Shib D. Banerjee , Bryan P. Toole (¶)

From the (1)Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Interactions of the glycosaminoglycan (GAG),()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.

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 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.

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.


EXPERIMENTAL PROCEDURES

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.

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 [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.

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).


RESULTS AND DISCUSSION

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.

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 [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.

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, 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.

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 -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 (Gluto 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.

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 -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.

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 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).

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.

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).

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)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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DE05838 and HD23681 (to B. P. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U20281 and U25026.

§
Present address: Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan.

To whom correspondence should be addressed. Tel.: 617-636-6659; Fax: 617-636-0380.

The abbreviations used are: GAG, glycosaminoglycan; bp, base pair; mAb, monoclonal antibody; nt, nucleotide; kb, kilobase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; IPTG, isopropyl-thiogalactoside.

Q. Yu, K. Deyst, E. Goedecke, S. D. Banerjee, and B. P. Toole, unpublished data.


ACKNOWLEDGEMENTS

We thank Al Sabbaj for technical assistance and Dr. Marion Gordon for helpful comments on the manuscript.


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