©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Forms of Mouse PG-M, a Large Chondroitin Sulfate Proteoglycan Generated by Alternative Splicing (*)

(Received for publication, June 21, 1994)

Kazuo Ito (§) Tamayuki Shinomura Masahiro Zako Minoru Ujita Koji Kimata (¶)

From the Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K.(1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.


INTRODUCTION

PG-M, a large chondroitin sulfate proteoglycan, is one of the major extracellular matrix molecules being expressed in various developing tissues as well as in several differentiated tissues such as smooth muscle in aorta(1) . This proteoglycan was for the first time isolated from chick limb buds at the prechondrogenic stages (2) and was shown to be expressed in the mesenchymal cell condensation area of chick limb buds(2, 3) . Such an expression pattern has suggested that PG-M may play some important roles as extracellular factors in actively differentiating tissues(1, 3) .

Recently, we have isolated cDNA clones encoding the entire core protein of chick PG-M(4) . The analysis of the deduced amino acid sequence revealed the presence of a hyaluronan-binding domain at the amino terminus and two epidermal growth factor (EGF)(^1)-like domains, a lectin-like domain and a complement regulatory protein-like domain at the carboxyl terminus. A human fibroblast chondroitin sulfate proteoglycan, versican, has been shown to have these structural characteristics and also have domains at both the amino terminus and the carboxyl terminus (5) with an extremely high identity to those of the PG-M core protein. Although the chondroitin sulfate attachment domain in the middle region of the PG-M core protein showed a low identity to the corresponding domain of versican, the finding of PG-M(V1), an alternatively spliced form of the PG-M core protein in the chondroitin sulfate attachment region, which was about 100 kDa smaller than the original form and similar in size to versican, has suggested that core proteins of various sizes are generated by alternative splicing in the chondroitin sulfate attachment region of the PG-M core protein and that versican could be a human equivalent of chick PG-M(V1)(4) .

We have shown that END-D cells derived from mouse aortic endothelium produce a large chondroitin sulfate proteoglycan capable of binding to hyaluronan, and judging from their structural and functional similarity we have concluded that the proteoglycan is identical or very closely related to chick PG-M(6) . Large aggregating chondroitin sulfate proteoglycans have recently been isolated from various tissues and cultured cells such as blood vessels(7, 8, 9) , brain(10) , aortic smooth muscle cells(11) , and skeletal muscle(12) . They also seem to be identical or closely related to PG-M or versican. In addition, by immunological analysis of chick PG-M core protein four core molecules different in size (550, 500, 450, and 300-350 kDa) were detected in chondroitin ABC lyase (EC 4.2.2.4) digested extracts of various embryonic chicken tissues(1) . Such heterogeneity of this molecule in structure as well as in distribution, together with the necessity not only to clarify the relationship between the multiple forms of PG-M and versican but also to examine the biological functions of PG-M by gene manipulation, has led us to perform cDNA analysis for the PG-M core proteins of mouse endothelial cells and subsequently those in a variety of other mouse tissues.

In the present study, we show a full-length cDNA sequence that encodes the core protein of a large chondroitin sulfate proteoglycan produced by mouse aortic endothelial cells. The analysis of the deduced amino acid sequence revealed that this molecule is one of the spliced forms of the PG-M core proteins corresponding to PG-M(V1)/versican. Northern blot analysis for mRNA species of various mouse tissues and subsequent sequencing of these transcripts suggest that a molecular form of PG-M may vary from tissue to tissue by alternative usage of at least two different exons (alpha and beta) encoding the chondroitin sulfate attachment region. A proposed designation for such a proteoglycan population and possible biological meanings of the heterogeneity by alternative splicing are discussed.


MATERIALS AND METHODS

Preparation and Screening of Mouse END-D Cell cDNA Library

Poly(A) RNA was extracted from cultured END-D cells with guanidine isothiocyanate (13) and purified by oligo(dT)-cellulose affinity chromatography. An oligo(dT)-primed cDNA library in gt11 was prepared by a You-Primed cDNA synthesis kit (Pharmacia Biotech Inc.). The library comprised 2.2 times 10^7 independent recombinants when transfected to Escherichia coli Y1088. A total of 1.0 times 10^6 independent clones were screened by plaque hybridization on Hybond N membrane (Amersham International plc, U. K.). The PCR product amplified from chicken PG-M cDNA (nucleotide positions 9901-10,698, corresponding to the C-terminal region) (4) was used as a probe for the first screening of this library. After the first cDNA clone was isolated, subsequent clones were picked up using 5`-ends of preceding cDNAs as probes. P labeling of cDNA probes was performed using a Random Primer DNA Labeling Kit (Takara, Kyoto, Japan).

Screening of Adult Mouse Brain cDNA Library

Adult mouse brain cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA), comprised 2.4 times 10^6 independent recombinants, was transfected to E. coli Y1088. A total of 8.0 times 10^5 independent clones were screened as described above using the P-labeled PCR product amplified from the inserted cDNA of m-Mg clone (nucleotide positions 716-1218, corresponding to a portion of the hyaluronan-binding domain, Fig. 1). The screening resulted in the isolation of two clones named m-Mo and m-Mq. Neither of them were hybridized with the P-labeled PCR product amplified from the inserted cDNA of m-Mh clone (nucleotide positions 1268-1820, corresponding to an N-terminal portion of the chondroitin sulfate attachment domain (beta) found in the END-D cell proteoglycan, Fig. 1). Subsequent clones were picked up using the 3`-ends of preceding cDNA clones as probes. DNA sequencing and homology analysis were performed as described below.


Figure 1: Comparative diagrams of isolated cDNA clones of mouse PG-M(V1) of cultured aortic endothelial cells and PG-M(V2) of adult mouse brain. Sizes and locations of the overlapping cDNA clones are indicated. The coding and noncoding regions are indicated by thick and thin lines, respectively. EcoRI restriction sites are indicated by arrowheads. The core protein structures are shown schematically. Domains of the coding region with high homology at both the amino-terminal and carboxyl-terminal regions are boxed and patterned. Chondroitin sulfate attachment domains (alpha and beta) are also boxed and patterned.



DNA Sequencing and Homology Analysis

Each inserted cDNA fragment of gt11 was subcloned into pGEM3Zf(-) plasmid vector (Promega Corp., Madison, WI). Nucleotide sequences were determined by the dideoxy chain termination method (14) using synthetic primers based on the 3`-end portions of the preceding sequences. Both strands of cloned cDNAs were sequenced. The obtained DNA sequences were compiled and analyzed using DNASIS computer programs (Hitachi Software Engineering Co., Japan). The deduced amino acid sequences were compared with other proteins in the data base compiled by the National Biomedical Research Foundation.

Northern Blot Analysis

Poly(A) RNAs were prepared from brain of 3-month-old adult mice (BALB/c) and limb buds of 11-14 day mouse embryos using the Fast Track mRNA isolation kit (Invitrogen Corp., San Diego, CA). About 2 µg of poly(A) RNA from cultured END-D cells, limb buds, and brain were electrophoresed in a denaturing formaldehyde-agarose gel (0.6%(w/v)). Blotting, prehybridization, and hybridization were performed as described previously(4) . Two different DNA probes used for hybridization were prepared by PCR amplification using isolated mouse cDNA clones as template. Probe A corresponds to the second tandem repeat portion (B` domain) of the hyaluronan-binding domain, and probe B corresponds to the beta domain of the chondroitin sulfate attachment region (see ``Results''). Their exact nucleotide positions are as follows: probe A, 927-1220; probe B, 5228-5738 (Fig. 2A). First hybridization was carried out with probe A. The membrane was then washed with 0.1 times SSPE containing 0.1% SDS at 65 °C. After it was exposed on x-ray film, the membrane was washed in boiling water containing 0.1% SDS to remove the first probe. The same membrane was again used for hybridization with probe B.



Figure 2: Nucleotide sequence and deduced amino acid sequence for mouse aortic endothelial cell PG-M core protein, PG-M(V1) (A), and those for adult mouse brain PG-M(V2) (B). The cDNA sequence for PG-M(V1) determined from the overlapping clones of Fig. 1together with the translation of an open reading frame of 2397 residues is shown in panel A. The nucleic acid and predicted amino acid sequence for the chondroitin sulfate attachment domain (alpha), which is unique to PG-M(V2) and contiguous to those of the amino- and carboxyl-terminal domains common to PG-M(V1), is shown in panel B. Consensus sequences for chondroitin sulfate attachment sites (acidic X-Ser-Gly, where X is one or two amino acids and either a hydrophobic or small neutral residue) proposed by Bourdon (18) are underlined. Alternative splicing sites are indicated by double underlines in the nucleotide sequence. Potential N-glycosylation sites are indicated by asterisks. Portions showing high identity with human versican in the chondroitin sulfate attachment domain (beta) of PG-M(V1) (amino acid positions 349-380 and 2061-2090 in panel A) are dotted underlined.



PCR Analysis

PCR analysis of PG-M cDNA was performed to detect cDNAs encoding different forms of the PG-M core protein in 11-14 day mouse embryonic limb bud cDNA library. (^2)PCR amplification was performed in a model PJ9600 DNA thermal cycler (Perkin-Elmer Cetus) using a GeneAmp PCR Reagent Kit (Takara, Kyoto, Japan) under the following conditions: 30 cycles at 95 °C for 1 min, 54 °C for 2 min, 72 °C for 3 min, and finally 72 °C for 15 min. The cDNA templates in each reaction were prepared from about 1 times 10^6 independent phage clones. The positions of primers used for PCR analysis are indicated as a and b in Fig. 5. Each primer was composed of an outer primer and an inner primer: Primer a, 5`-CAGCCAACAAGACCATCAG-3`(4062-4080) and 5`-CTACTTCAACACCTGCA-3`(4120-4136); Primer b, 5`-TTCCCATTGATATACTGCACTG-3`(1437-1458) and 5`-ACGGAGTAGTTGTTACATCCG-3`(1416-1436). The PCR was performed by combinations of the outer primers for the first amplification and inner primers for the second. Amplified products were analyzed by agarose gel electrophoresis.


Figure 5: PCR analysis and the sequences at the alternatively spliced sites of PG-M(V0). A, the positions of specific primers used for PCR analysis are indicated as a and b. Domains at both the amino- and carboxyl-terminal regions and the chondroitin sulfate attachment domains (alpha and beta) at the central region are boxed and patterned as shown in Fig. 1. B, the PCR products are analyzed by agarose gel electrophoresis. DNA size markers are shown at the right. C, the PCR product from mouse limb bud cDNA library was sequenced, and the part of the sequence around the alternative splicing site was shown. The nucleotide positions for the alternative splicing sites are indicated by arrows, and both the nucleotide positions and sequences for the chondroitin sulfate attachment domain alpha are shown in boldface type.




RESULTS

Isolation and Sequencing of PG-M cDNA Clones from Cultured Mouse Endothelial Cells

Taking advantage of an extremely high homology of the carboxyl-terminal region of chicken PG-M core protein to the one of human versican(4) , the first cDNA clone was isolated from an END-D cell cDNA library by hybridization with a PCR product amplified from the 3`-region of chicken PG-M cDNA (nucleotide positions 9901-10,698, corresponding to the carboxyl-terminal regions of the PG-M core protein). After the first cDNA clone was isolated, subsequent clones were picked up using the 5`-terminal regions of preceding cDNA clones as probes. A total of seven clones thus obtained covered the entire coding sequence of the PG-M core protein expressed by cultured mouse endothelial cells (Fig. 1).

The composite sequence is 7547 nucleotides long and encodes 2397 amino acids ( Fig. 1and Fig. 2A), which suggests that this isolated cDNA may correspond to chicken PG-M(V1), a short one of the alternatively spliced forms of the PG-M core protein (4) or human versican(5) . Included in this sequence are 178 nucleotides of 5`-leader and 175 nucleotides of 3`-trailer sequence. The nucleotide sequence immediately upstream from the ATG codon is in good agreement with the consensus sequence for translational initiation in eukaryotes(16) . The putative signal peptide sequence consists of the amino-terminal 20 amino acid residues with a putative cleavage site between Ala-20 and Leu-21. This site is in agreement with the(-3, -1) rule of von Heijne(17) . There are a total of 36 cysteine residues in the core protein, of which 22 residues are in the carboxyl-terminal portion, 12 residues are in the amino-terminal portion, and 2 residues are in the amino-terminal side of the chondroitin sulfate attachment region. Different from chicken and human molecules, one extra cysteine residue is present at the amino-terminal region. There are a total of 31 potential chondroitin sulfate attachment sites at the middle part of the core protein, that have Ser-Gly or Gly-Ser sequences. Eleven of them are in good agreement with the consensus sequence of chondroitin sulfate attachment sites, acidic X-Ser-Gly proposed by Bourdon(18) . In addition, there are nine potential N-glycosylation sites (19) and 45 potential threonine-O-glycosylation sites(20) . Both types of glycosylation sites are distributed almost uniformly on the core protein.

As was expected of mouse PG-M, the deduced amino acid sequence for the core protein revealed the presence of a link protein-like sequence (corresponding to the hyaluronan-binding domain) at the amino-terminal region and two EGF-like sequences, a lectin-like sequence, and a complement regulatory protein-like sequence at the carboxyl-terminal region. In addition, those domains of this mouse proteoglycan show an extremely high identity to corresponding domains of chicken and human molecules (Fig. 3), particularly at the carboxyl-terminal regions. Of the two EGF-like domains consisting of 76 amino acid residues, the second domain consisting of 39 amino acid residues was completely identical in amino acid sequences between mouse and human. Further, in the lectin-like domain composed of 129 amino acid residues, only 1 residue was different between mouse and human, and 5 residues between mouse and chicken. In the complement regulatory protein-like domain composed of 61 amino acid residues, only 1 residue was different between mouse and human, and 3 residues were different between mouse and chicken. Cysteine residues in these domains were all at the same positions among three animals.


Figure 3: Comparisons of amino acid sequences of the amino-terminal region and the carboxyl-terminal region. The same amino acid residues among human, mouse, and chick are shaded. Cysteine residues are shown in boldface type, and one residue specifically found in mouse PG-M is indicated by an asterisk. The data of human versican are taken from (5) .



In contrast, such a high identity was not seen in the chondroitin sulfate attachment region at the middle part of the core protein. In addition, based on a comparison of molecular weights estimated from deduced amino acid sequences the region was about 100 kDa smaller than that of chicken PG-M core protein (3562 amino acid residues) and appeared to correspond to chicken PG-M(V1) or human versican. Thus, a comparison of the amino acid sequence of the chondroitin sulfate attachment region was made among these molecular species. Some identity was observed between mouse and human (52.8%), and essentially no identity was observed between mouse and chicken (21.7%). Interestingly, about 30 amino acid residues at both the starting and ending portions of this region (see Fig. 2A) showed a high identity (about 80%) between mouse and human.

Northern Blot Analysis

The above results showing that mouse endothelial cells synthesize PG-M(V1), a short form of PG-M, have suggested a tissue-dependent heterogeneity of PG-M core protein transcripts caused by alternative splicing. To test this possibility, poly(A) RNAs were prepared from limb buds and adult brain as described under ``Materials and Methods.'' Northern blot analysis was first performed using probe A shown in Fig. 4(top). The hybridization of Poly(A) RNA samples from these tissues and cultured END-D cells revealed the presence of at least six mRNA species of different sizes (Fig. 4, lanes 1, 3, and 5). Each approximate size is 12, 10, 9, 8, 7.5, and 6.5 kb long, respectively. In the END-D cell sample, there were three distinct bands of 10, 9, and 8 kb in size (Fig. 4, lane 1). In the embryonic limb buds, a 12-kb mRNA band was detected in addition to three bands of 10, 9, and 8 kb in size (Fig. 4, lane 5). On the other hand, in the adult brain only 7.5- and 6.5-kb mRNA bands were detected (Fig. 4, lane 3). There were smearing bands of less than 4.7 kb in size. Since smearing bands of less than 4.7 kb in size often varied in intensity, depending upon a batch difference of the same tissue samples, they might be caused by some artificial degradation of the mRNAs, and/or co-migration of contaminant ribosomal RNAs might cause the abnormal migration. However, it is still likely that other smaller forms of PG-M or some molecules closely resembling PG-M may exist.


Figure 4: Northern blot analysis of mRNAs for PG-M core proteins in various mouse tissues. Poly(A) RNA samples from cultured END-D cells (lanes 1 and 2), adult brain (lanes 3 and 4), and embryonic limb buds (lanes 5 and 6) were electrophoresed and transferred to a Hybond N membrane. The bound RNAs were first hybridized with probe A (lanes 1, 3, and 5) and then hybridized with probe B (lanes 2, 4, and 6) as described under ``Materials and Methods.'' The location of each probe is shown at the top. Positions of RNA molecular size markers are indicated in kilobases at the right. Six mRNA bands in different sizes are shown by arrows at the left.



It was of note that probe B shown in Fig. 4(top) was hybridized to the four mRNA bands of 12, 10, 9, and 8 kb in size from END-D cells and limb buds sample (Fig. 4, lanes 2 and 6) but not to the smaller two bands of 7.5 and 6.5 kb in size from adult brain sample (Fig. 4, lane 4). Since probe B is for the chondroitin sulfate attachment domain derived from one large exon of 5.2 kb, (^3)the result suggests that the 7.5- and 6.5-kb PG-M transcripts may have the different chondroitin sulfate attachment regions generated by alternative usage of different exons encoding the chondroitin sulfate attachment domains, which was expected from the difference in the chondroitin sulfate attachment region found between chicken PG-M and PG-M(V1)(4) .

Sequencing of the Second Chondroitin Sulfate Attachment Domain (alpha) in the 7.5- and 6.5-kb Transcripts

To investigate the above possibility, we obtained cDNA clones for these short PG-M transcripts as described under ``Materials and Methods.'' Screening of adult mouse brain cDNA library resulted in the isolation of three clones, which covered the entire coding sequence of the above transcripts (Fig. 1). Sequencing and homology analysis have revealed a deduced protein structure as shown in Fig. 1. The composite sequence encodes 1614 amino acids. As was expected, the nucleotide sequences corresponding to the amino- and carboxyl-terminal regions are completely identical to those of mouse endothelial cell PG-M described above. The nucleotide and amino acid sequences of the chondroitin sulfate attachment domain at the middle portion of the core protein (CS alpha) shown in Fig. 2B neither showed homology to the corresponding domain of mouse endothelial cell PG-M, nor to the other chondroitin sulfate attachment domain of chicken PG-M that is spliced out when PG-M(V1) is generated. As shown in Fig. 6B, this domain has no cysteine residue but has a total of 13 potential chondroitin sulfate attachment sites, of which three sites are in good agreement with the consensus sequence(18) . In addition, there are six potential N-glycosylation sites (19) and 25 potential O-glycosylation sites(20) . The results suggest that the small transcripts of 7.5 and 6.5 kb in size in adult mouse brain may correspond to one of the alternatively spliced forms in that they are different from other transcripts in having the smaller chondroitin sulfate attachment domain (CS alpha) than PG-M(V1) or versican. We here designate these transcripts as PG-M(V2) (Fig. 6A).


Figure 6: Schematic presentation of the relationship among different PG-M transcripts (A) and predicted structure of PG-M(V0) (B). A, domains at both the amino- and carboxyl-terminal regions and the chondroitin sulfate attachment domains (alpha and beta) at the central region are boxed and patterned as shown in Fig. 1. B, locations of chondroitin sulfate attachment sites (Ser-Gly or Gly-Ser sequences), N-glycosylation sites, and cysteine residues are shown by vertical lines, respectively. Consensus sequences for chondroitin sulfate attachment sites proposed by Bourdon (18) are indicated by thick vertical lines. One cysteine residue specifically found in mouse PG-M is indicated by an arrowhead.



PCR Analysis for a Transcript without the Alternative Splicing

Thus, it is likely that alternative splicing of the primary gene of PG-M yielded those multiple transcripts by splicing out either of the two different exons for the chondroitin sulfate attachment domains (splicing out CS alpha domain for PG-M(V1) or CS beta domain for PG-M(V2)) or by including both. The largest transcript of the PG-M core protein is now designated as PG-M(V0), which is a transcript without the alternative splicing and contains both CS alpha and beta domains. In chicken, this PG-M(V0) form has been identified as a 13-kb transcript by Northern blot analysis of the limb bud sample (4) . In mouse, a 12-kb mRNA band detected in the limb bud sample might correspond to the PG-M(V0) form (Fig. 4, lanes 5 and 6). Therefore, we examined whether or not the PCR analysis could detect a transcript containing both the chondroitin sulfate attachment domains (alpha and beta). For this purpose, we used the following combination of oligonucleotide primers a and b, flanking the putative splicing site. The reaction yielded a 297-base pair product from mouse limb bud cDNA library (Fig. 5B). The nucleotide sequence of this product suggested the presence of transcript corresponding to PG-M(V0) (Fig. 5C).

The relationship among three transcripts for the different forms of the PG-M is shown in Fig. 6A, and schematic presentation of the PG-M(V0) structure including putative chondroitin sulfate attachment sites, putative N-glycosylation sites, and the locations of cysteine residues is in Fig. 6B.


DISCUSSION

Earlier studies including ours on proteoglycans synthesized by stage 22-23 chick embryo limb buds revealed the occurrence of a unique band with a sedimentation velocity that was different from those of proteoglycans found in differentiated cartilage. This component was referred to as Fraction III(21) , PCS-M(22) , Fraction II(23) , or PGS(LM)-1(24) . In 1986 we for the first time isolated and characterized the proteoglycan and named it PG-M because of the predominant proteoglycan from the mesenchyme(2) . Since then, we have continued to use this designation in our studies on functions and distribution of PG-M(1, 3, 4, 25, 26, 27) . In addition, we have shown from cDNA study on PG-M from chick limb buds that versican may only correspond to one of the alternatively spliced forms of the PG-M core protein(4) . We therefore continue to use this designation, although the name of versican has become popular in any event.

In the present study we have shown a full-length cDNA sequence of mouse PG-M and suggested that alternative splicing of a primary PG-M gene transcript generates multiforms of the core protein. As shown in Fig. 6A we have demonstrated the occurrence of at least three different forms of the PG-M core protein caused by alternative usage of two different exons encoding chondroitin sulfate attachment region (CS alpha and beta domains). We have proposed the designation for each transcript of the PG-M core protein as follows: PG-M(V0), the transcript (12 kb) without the alternative splicing; PG-M(V1), the larger ones (10, 9, and 8 kb) of the alternatively spliced forms, which have the beta domain of the chondroitin sulfate attachment region and correspond to human versican; PG-M(V2), the smaller ones (7.5 and 6.5 kb) of the alternatively spliced forms, which have the alpha domain of the chondroitin sulfate attachment region.

The present study could not answer the question how the size differences were generated within 10-, 9-, and 8-kb transcripts and within 7.5- and 6.5-kb transcripts. The similar heterogeneity in size was observed with versican in human osteogenic sarcoma cells(28) . A rapid amplification of cDNA ends at the 5`-terminus did not yield 1- or 2-kb size difference among them.^3 Aggrecan expressed by human cartilage has been reported to undergo alternative splicing of the EGF-like domain at the carboxyl-terminal G3 domain(29) . We therefore examined whether or not the alternative splicing might also occur at the amino-terminal and/or carboxyl-terminal regions of PG-M core protein to cause such heterogeneity of transcripts. PCR amplification using several mouse cDNA libraries and combinations of appropriate primers did not show the possibility (yielded only a single band).^3 Thus, the heterogeneity of 1- or 2-kb size might be caused by some differences of the noncoding region at the 3`-end. Alternatively, it is also possible that this size difference of transcripts might be caused by some artificial degradation.

Although chick PG-M(V1) probably corresponds to human versican, the chondroitin sulfate attachment region of chick PG-M(V1) is still about 700 nucleotide residues longer than that of human versican or mouse PG-M(V1). The difference might be caused by a species specificity because of no detection on our trial basis of further alternative splicing in the chondroitin sulfate attachment region of PG-M(V1) (data not shown). Further analysis of the PG-M genomic gene would be needed to reveal their relationship.

In the present study, expression of PG-M transcripts of various sizes was identified in embryonic limb buds, in adult brain, and in cultured aortic endothelial cells. The results may suggest the tissue-dependent regulation of the alternative splicing. Consistent with these observations, immunological analysis for PG-M core proteins of various chick tissues suggested previously that there were at least four core molecules with different sizes (550, 500, 450, and 300-350 kDa) and their appearance varied in a tissue-dependent manner(1) . For example, all of these four core molecules were detected in embryonic aorta and lung, a 450-kDa core molecule in embryonic skeletal muscle, and a 300-350-kDa molecule in embryonic brain. In brain, existence of several smaller sized core molecules was also suggested. Our present study has suggested that alternative splicing of mRNA may cause such a multiplicity of PG-M core protein, although some may be derived from undergoing posttranslational modification as well. In vitro translation of various PG-M mRNA species and analysis of products would clarify their relationship. In addition, in situ hybridization with appropriate probes would reveal molecular forms of mRNAs expressed in certain tissues at certain stages.

Neurocan and brevican have recently been cloned and identified as chondroitin sulfate proteoglycans unique to brain(30, 31) . Their structures resemble those of PG-M, versican, and aggrecan with regard to the presence of the same domain elements at the amino- and carboxyl-terminal regions. The coding regions of the cDNAs are about 3.8 and 2.2 kb in size, and the mRNAs are about 7.5 and 3.3 kb in size for rat neurocan and bovine brevican, respectively. Considering these sizes, PG-M(V2) might be a mouse equivalent of rat neurocan. The comparison in amino acid sequences of the amino- and carboxyl-terminal domains between mouse PG-M(V2) and rat neurocan showed about 50% identity at the amino-terminal domains and 57% at the carboxyl-terminal domains. In contrast to the extremely high conservation of these domains of PG-M among different animal species, the identity between mouse PG-M(V2) and rat neurocan is rather low. Bovine brevican is distinctly shorter in size than PG-M(V2). In addition, the comparison between mouse PG-M(V2) and bovine brevican showed even to the highest degree a 64% identity at the amino-terminal B domain and a 61% identity at the carboxyl-terminal lectin-like domain. Taken together, these data indicate that PG-M of smaller sizes may be a different molecule from neurocan and brevican.

Some extracellular matrix proteins such as fibronectin, tropoelastin, tenasin, collagen (types II, VI, and XIII), and CD44 have been reported to have a diversity of molecular forms generated by alternative exon usage(32) . Several isoforms of these proteins have been shown to be regulated developmentally and/or in tissue-dependent manners(32) . Functional significance of alternative splicing has been extensively analyzed in fibronectin, and form-specific functions related to regulation of dimer formation, secretion(33) , cell adhesiveness(34) , and incorporation into fibrin clots (35) have been identified. The function of the alternatively spliced region of CD44 has been shown to reflect the ability to adhere to lymph node stromal cells(15) . Our present study has shown that the alternative usage of two different exons encoding chondroitin sulfate attachment domains may yield at least three different forms of PG-M with different sizes of the chondroitin sulfate attachment region. It has been shown in our laboratory that PG-M had a strong inhibitory effect on the adhesion of various types of cells to substrate-adhesive glycoproteins, and the attached chondroitin sulfate chains were responsible for this activity (26) . The finding emphasized the importance of chondroitin sulfate chains in the proteoglycan as an inhibitory modifier in the regulation of cell-substrate interactions and, therefore, suggests that alternative splicing in the chondroitin sulfate attachment domain may be related to one of important cellular mechanisms operative for the regulation of cell-matrix interactions during cell differentiation and tissue morphogenesis.


FOOTNOTES

*
This work was supported in part by special coordination funds of the Science and Technology Agency of the Japanese Government; a grant-in-aid from the Ministry of Education, Culture, and Science of the Japanese Government; and a special research fund from Seikagaku Corporation. 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(TM)/EMBL Data Bank with accession number(s) D16263 [GenBank]and D28599[GenBank].

§
Present address: Dept. of Orthopedic Surgery, Sapporo Medical University, S1-W16, Chuo-ku, Sapporo 060, Japan.

To whom all correspondence should be addressed. Tel.: 81-52-264-4811; Fax: 81-56-163-3532.

(^1)
The abbreviations used are: EGF, epidermal growth factor; PCR, polymerase chain reaction; kb, kilobase pairs; bp, base pairs.

(^2)
M. Ujita, T. Shinomura, and K. Kimata, submitted for publication.

(^3)
T. Shinomura, K. Ito, M. Zako, M. Ujita, and K. Kimata, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Dr. S. Ishii (Sapporo Medical University), Dr. H. Iwata (Nagoya University), and Dr. N. Katsura (Nagasaki University) for continuous support and encouragement. We are also grateful to Drs. G. Eguchi and T. Takeuchi (National Institute for Basic Biology) for providing END-D cells.


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