©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of PG-M(V3), an Alternatively Spliced Form of PG-M without a Chondroitin Sulfate Attachment Region in Mouse and Human Tissues (*)

(Received for publication, September 23, 1994; and in revised form, November 30, 1994)

Masahiro Zako Tamayuki Shinomura Minoru Ujita Kazuo Ito 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 showed previously that the alternative splicing of chondroitin sulfate attachment domains (CS alpha and CS beta) yielded multiforms of the PG-M core protein in mouse. A transcript encoding a new short form of the core protein PG-M(V3) was found in various mouse tissues using polymerase chain reaction. DNA sequences of the polymerase chain reaction products suggested that PG-M(V3) had no chondroitin sulfate attachment domain. PG-M(V3) was also detected in various human tissues. The presence of a transcript for PG-M(V3) was further supported by Northern blot analysis. Southern blot analysis confirmed that multiforms of the PG-M core protein, including PG-M(V3), were derived from a single genomic locus by an alternative splicing mechanism. Because PG-M(V3) has no chondroitin sulfate attachment region, which is the most distinctive portion of a proteoglycan molecule, this form may have a unique function.


INTRODUCTION

PG-M, a large chondroitin sulfate proteoglycan, is one of the major extracellular matrix molecules in the mesenchymal cell condensation regions of developing limb buds(1) . The expression, however, is regulated in an inverse relationship to that of aggrecan, and PG-M disappears after the cartilage development(2) . Such a transient expression of this proteoglycan is also seen in various embryonic tissues during morphogenesis and differentiation(3) . Therefore PG-M can be expected to play some regulatory roles in such biological events.

Our recent cDNA studies on the core proteins of PG-M in embryonic chick limb buds (4) and mouse aortic endothelial and brain cells (5) have revealed that six different mRNA species are expressed in a tissue-dependent manner and that at least three different core proteins are encoded by them (designated PG-M(V0), PG-M(V1), and PG-M(V2) in order of length). All have both the hyaluronan-binding domains at the amino terminus and two epidermal growth factor (EGF(^1))-like domains, a lectin-like domain, and a complement regulatory protein (CRP)-like domain at the carboxyl terminus. However, they are different in the chondroitin sulfate attachment regions in the middle of the core proteins in that the differences are generated by alternative and simultaneous usages of the two different domains for the chondroitin sulfate attachment region (CS alpha and CS beta).

Versican, a fibroblast proteoglycan, was first identified by the cDNA study(6, 7) . Homology analysis of the deduced amino acid sequence demonstrated that versican corresponded to PG-M(V1) of the chicken or mouse PG-M core protein(5) . By comparison of the amino acid sequences among three animal species, an extremely high homology is observed with both the amino-terminal and carboxyl-terminal regions but not with the chondroitin sulfate attachment region in the middle of the core protein (5) . The simultaneous presence of such evolutionally conserved and non-conserved structures in the PG-M core protein might have some important biological meanings. Such differences suggest that the amino- and carboxyl-terminal regions and the chondroitin sulfate attachment region of the PG-M core protein may have different functions and that the alternative splicing in the latter region may be related to the function of the chondroitin sulfate attachment region(5) . Consistent with this possibility, we showed previously that the chondroitin sulfate chains in the proteoglycan are the active sites for the inhibitory activity of PG-M in the regulation of cell to substrate interactions (8) and that the activity per molecule might vary depending on the contents of the chains per molecule due to the alternative splicing(5) . On the other hand, the amino- and carboxyl-terminal regions showed the binding activity to hyaluronan (1, 9) and a C-type lectin-like activity(10) , respectively, and might participate in the appropriate localization of the proteoglycan in the extracellular matrix mesh work via the bindings to hyaluronan and sugar-containing molecules(3, 5) .

In the present study, we demonstrate the occurrence of a novel population of PG-M(V3) in some mouse and human tissues that do not have any chondroitin sulfate attachment region with an alternative splicing mechanism.


MATERIALS AND METHODS

cDNA Libraries

Poly(A) RNA was isolated from limb buds of day 11-14 mouse embryos by the guanidine isothiocyanate method(11) , followed by Oligotex-dT30 super (Japan Roche, Tokyo, Japan). An oligo(dT)-primed cDNA library was constructed in gt11 as described previously(12) . Preparation of a mouse END-D cell cDNA library was as described previously(5) . Adult mouse brain cDNA library, adult mouse skeletal muscle cDNA library, adult human cerebral cortex cDNA library, fetal human liver cDNA library, and adult human stomach cDNA library were obtained commercially (CLONTECH, Palo Alto, CA).

Detection of PG-M(V3) in Various Mouse Tissues by Polymerase Chain Reaction (PCR)

Four different mouse cDNA libraries were examined by the PCR analysis: brain cDNA library, END-D cell cDNA library, limb bud cDNA library, and skeletal muscle cDNA library.

PCR amplifications were performed with a DNA thermal cycler, model PJ 9600 (Cetus Co., Emeryville, CA), using a GeneAmp PCR reagent kit (Takara Biomedicals, Kyoto, Japan) and Perfect Match polymerase enhancer (Stratagene, La Jolla, CA) for 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. Sequences and positions of specific primers used for the PCR analysis are indicated in Fig. 2and Table 1. The first PCR amplification was performed using a pair of the outer primers (see Fig. 2, a and d), and then the second PCR amplification was performed on the first PCR amplification products as templates using a pair of the inner primers (see Fig. 2, b and c). The final products were analyzed by agarose gel electrophoresis on NuSieve 3:1 (FMC Corp. BioProducts, Rockland, ME).


Figure 2: PCR analysis for PG-M(V3) in various mouse cDNA libraries. Positions of outer and inner combinations of primers used for the PCR analysis are indicated by arrowheads (a and d for the outer, b and c for the inner) on the schematic diagram. PCR products were derived from the following four different mouse cDNA libraries: brain (lane 1), END-D cell (lane 2), limb bud (lane 3), and skeletal muscle (lane 4). They were analyzed by agarose gel electrophoresis. DNA size markers are shown on the right. HABR, hyaluronan-binding region; LEC, lectin.





DNA Sequencing of PCR Products

The second set of PCR-amplified products was purified from the agarose gel by Geneclean II (BIO 101 Inc., La Jolla, CA) and subjected to direct sequencing using an AmpliTaq cycle sequencing kit (Perkin-Elmer)(13) . The inner primers used for the second PCR reaction were radiolabeled at the 5`-end with [-P]ATP (specific radioactivity, 6000 Ci/mmol) (Amersham International plc), using T(4) polynucleotide kinase (Takara Biomedicals) and used for the sequencing.

Northern Blot Analysis

Mouse END-D cell poly(A) RNA was isolated by a guanidine isothiocyanate method (11) followed by oligo(dT)-cellulose affinity chromatography. Human retina poly(A) RNA (whole retina of adult) and human brain poly(A) RNA (whole cerebral brain of adult) were obtained commercially (Stratagene).

7 µg of mouse END-D cell poly(A) RNA, 5 µg of human retina poly(A) RNA, and 5 µg of human brain poly(A) RNA were electrophoresed in a denaturing formaldehyde-agarose gel (0.8%) and transferred to a Hybond N membrane (Amersham International plc) by a vacuum blotter, VacuGene XL (Pharmacia Biotech Inc.), under alkaline blotting conditions as recommended by the manufacturer.

Three different probes were used for hybridization (see Fig. 4). A 50-bp oligonucleotide (5`-GGGTTTGTTTTGCAGAGATCAGGTCGTTTAAAGCAGTAGGCATCAAATCT-3`) corresponding to nucleotide positions 1196-1245 of mouse PG-M(V3) and a 32-bp oligonucleotide (5`-TTGCAGCGATCAGGTCGTTTAAAGCAGTAGGC-3`) corresponding to nucleotide positions 1132-1163 of human PG-M(V3) were radiolabeled at the 5`-end with [-P]ATP as described above and used as probe A and C, respectively. A 338-bp cDNA fragment corresponding to nucleotide positions 284-621 (hyaluronan-binding domain) of mouse PG-M(V3) was radiolabeled with [alpha-P]dCTP by the random priming method (14) and was used as probe B. Prehybridization and hybridization were performed at 42 °C in the presence of 50% (v/v) formamide and 10% (w/v) dextran sulfate for probe B for 24 h as described previously(4, 15) . For probe A and C, prehybridization and hybridization were performed without formamide and dextran sulfate. The membrane was then washed with 0.1 times SSPE (18 mM NaCl, 1.0 mM sodium phosphate, 1.0 mM EDTA) containing 0.1% (w/v) SDS at 62 °C for probe B and 1 times SSPE (180 mM NaCl, 10 mM sodium phosphate, 10 mM EDTA) containing 0.1% (w/v) SDS at room temperature for probe A and C and exposed on x-ray film (Fuji-RX, Fuji Photo Film Co.). Sizes of RNA were determined using an RNA ladder (Life Technologies, Inc.).


Figure 4: Northern blot analysis of transcripts encoding mouse and human PG-M(V3). Mouse END-D cell poly(A) RNA (lanes 1 and 2), human retina poly(A) RNA (lane 3), and human brain poly(A) RNA (lane 4) were separated on denaturing formaldehyde-agarose gels. After transfer to a Hybond N membrane, the bound mRNAs were hybridized with probe A (lane 1), B (lane 2), and C (lanes 3 and 4). Locations of probes are shown on the schematic diagram. Sizes of the molecular markers for calibration are indicated on the right in kilobases. HABR, hyaluronan-binding region; LEC lectin.



Genomic Southern Blot Analysis

Mouse genomic DNA was obtained from CLONTECH. Five micrograms of the DNAs were digested with either SphI, SacI, KpnI, HindIII, HindII, BglII, or BamHI. The digests were electrophoresed in a 0.7% (w/v) agarose gel and then transferred to a Hybond N membrane by a vacuum blotter as described above. The membrane was hybridized with P-labeled probe B used for the above Northern hybridization (nucleotide position 284-621). Prehybridization and hybridization were done at 65 °C using Rapid hybridization buffer (Amersham International plc), followed by washes and autoradiography as described above.

Detection of PG-M(V3) in Various Human Tissues by PCR

Sequences of primers were chosen based on the published sequences of versican/human PG-M(V1) (see Fig. 6and Table 1, primers e-p), and the primers were prepared as described above. Adult cerebral cortex cDNA library, adult stomach cDNA library, and fetal liver cDNA library were used as a template. PCR amplification and subsequent characterization of PCR products were performed as described above.


Figure 6: PCR analysis for PG-M(V3) in various human cDNA libraries. PCR amplification was performed on the junction region from the amino-terminal side (A), from the carboxyl-terminal side (B), and in the middle (C) using various combinations of primers. Positions of outer and inner combinations of primers used for the PCR analysis are indicated by arrowheads on the schematic diagram. (A, primers e and h for the outer and primers f and g for the inner at the amino-terminal side; B, primers i and l for the outer and primers j and k for the inner at the carboxyl-terminal side; C, primers m and p for the outer and primers n and o for the inner at the middle part.) PCR products were derived from the following human tissues and combinations of primers: lanes 1 and 2, adult cerebral cortex cDNA library using combinations of primers indicated in A and B, respectively; lane 3, adult stomach cDNA library using combinations of primers indicated in C; and lane 4, fetal liver cDNA library using combinations of primers indicated in C. Combinations of primers indicated in A and B yielded no PCR product from stomach and fetal liver cDNA libraries, probably due to the absence of cDNAs covering those regions (data not shown). Nucleic acid and predicted amino acid sequences of PCR products (308 bp) from the human adult stomach and fetal liver cDNA libraries are shown in Fig. 3B. DNA size markers are shown on the right. Composite restriction endonuclease sites of the human PG-M(V3) core protein that were used to characterize the products are also shown. HABR, hyaluronan-binding region; LEC, lectin.




Figure 3: Nucleic acid and predicted amino acid sequences of products amplified by PCR. A, a 341-bp product from mouse END-D cell cDNA library. B, 308-bp products from the human adult stomach and fetal liver cDNA libraries. Triplet nucleotide codes, which are composed of both a hyaluronan-binding domain and an EGF-like domain, are underlined. Predictable splicing points are indicated by arrows.




RESULTS

Expression of PG-M(V3) in Various Mouse Tissues and Cells

We have shown the occurrence in mice of at least three different forms of the PG-M core protein generated by alternative splicing of the chondroitin sulfate attachment region (CS domains alpha and beta) (Fig. 1)(5) . Each form was designated in order of the length: PG-M(V0) for the largest size without the alternative splicing; PG-M(V1) for the second largest of the alternatively spliced forms having the CS attachment domain beta and corresponding to versican; and PG-M(V2) for the third largest of the alternatively spliced forms having the CS attachment domain alpha. It should be noted that all three forms had a hyaluronan-binding domain at the amino termini and two EGF-like domains, a lectin-like domain, and a CRP-like domain at the carboxyl termini.


Figure 1: Alternatively spliced multiforms of mouse PG-M core protein, PG-M(V0), PG-M(V1), PG-M(V2), and PG-M(V3). Hyaluronan-binding domain, EGF-like domain, lectin-like domain, and CRP-like domain are all present in those forms. Chondroitin sulfate attachment domains at the middle region are regulated by alternative splicing. PG-M(V1) is a mouse equivalent of human versican. The number of amino acids (aa) is indicated at the top.



Those structural characteristics made us expect the possibility that an alternatively spliced form without the CS attachment domain might occur in some tissues. We have examined this possibility by the PCR amplification method on mouse tissues and cells taking advantage of the known sequence (Fig. 2). The PCR primers were chosen from the hyaluronan-binding domain at the amino terminus and the EGF-like domain at the carboxyl terminus. An expected 341-bp product was amplified from both the brain cDNA (Fig. 2, lane 1) and END-D cell cDNA libraries (Fig. 2, lane 2). However, it could not be detected in limb bud cDNA library (Fig. 2, lane 3) or in skeletal muscle cDNA library (Fig. 2, lane 4).

To confirm that the PCR product was derived from a postulated form of PG-M without the CS attachment domains, the DNA sequence of a PCR product from the mouse END-D cell cDNA library was determined. A 341-bp product had the completely expected sequence (Fig. 3A). Neither a new termination codon nor a shift of reading frame was found in this sequence. Therefore, it is likely that some mouse tissues produce a PG-M-like molecule that has the amino- and carboxyl-terminal regions identical to those of the other PG-M forms but lacks the CS attachment region. So we termed this molecule PG-M(V3). We obtained PCR products covering the entire PG-M(V3) from the mouse END-D cell cDNA library and determined the sequence. The whole nucleotide sequence encoding mouse PG-M(V3) has been submitted to the GenBank/EMBL Data Bank with accession number D32040.

The presence of mRNA encoding PG-M(V3) in mouse tissues was further supported by Northern blot analysis (Fig. 4). A 3-kb mRNA was detected in mouse END-D cell by hybridization with probe A that encoded the sequence for the junction of PG-M(V3) (Fig. 4, lane 1). The 3-kb mRNA was also hybridized with probe B that encoded the hyaluronan-binding domain of PG-M (Fig. 4, lane 2). Therefore, this transcript must correspond to PG-M(V3). Other transcripts hybridized with probe B corresponded to the other forms of PG-M described previously(5) . Because 0.8% agarose gel was used for the electrophoretic analysis to detect transcripts of about 3 kb encoding mainly PG-M(V3), it was difficult to assess the exact sizes of the larger transcripts. Also co-migration of contaminant ribosomal RNAs might have caused the abnormal migration. Therefore, precise comparison of the sizes and amounts of those transcripts should not be made with our previous results on END-D cells(5) .

Southern Blot Analysis

Southern blot analysis of mouse genomic DNA was performed to verify whether PG-M(V0), PG-M(V1), PG-M(V2), and PG-M(V3) were derived from a single genomic locus by the alternative splicing mechanism. Hybridization was performed using probe B encoding the common sequence in all PG-M forms described above in the Northern blot analysis. A single band was detected in each of the seven digests (Fig. 5), although five bands have been detected in Northern blot analysis using the same probe (Fig. 4, lane 2). These results indicate that PG-M(V0), PG-M(V1), PG-M(V2), and PG-M(V3) are derived from the same genomic locus by an alternative splicing mechanism.


Figure 5: Southern blot analysis of mouse genomic DNA encoding PG-M core protein. Mouse genomic DNAs were digested with seven different restriction enzymes indicated at the top and then were separated in a 0.7% agarose gel. After transfer to a Hybond N membrane, the bound DNAs were hybridized with a P-labeled probe B as shown in Fig. 4. Sizes of the DNA species used for calibration are indicated on the right in kilobases.



Expression of PG-M(V3) in Human Tissues

We then examined PG-M(V3) in human tissues. PCR analysis was performed to detect the occurrence of PG-M(V3) in various human tissues (Fig. 6). Almost the full length of the cDNA was detected using two primer sets in the cerebral cortex cDNA library, one for the amino terminus (Fig. 6A) and another for the carboxyl terminus (Fig. 6B), both of which strided over the junction. The expected sizes of products were detected as 1183 bp for the amino terminus and 959 bp for the carboxyl terminus, respectively (Fig. 6, lane 1 and lane 2, respectively). Furthermore, another primer set (Fig. 6C) yielded 308-bp products from the adult stomach cDNA library (Fig. 6, lane 3) and the fetal liver cDNA library (Fig. 6, lane 4). DNA sequences of the 308-bp products from the adult stomach and fetal liver cDNA libraries were determined as described above. The products completely matched the expected sequences (Fig. 3B). There was also neither the termination codon nor the shift of reading frame. Restriction enzyme mappings of the PCR products (Fig. 6, A and B) were consistent with the sequences of PG-M(V3). We obtained PCR products covering the entire PG-M(V3) from the human cerebral cortex cDNA library and determined their sequences. The whole nucleotide sequence encoding human PG-M(V3) has been submitted to the GenBank/EMBL Data Bank with accession number D32039.

The occurrence of PG-M(V3) in human tissues was further confirmed by Northern blot analysis. A short transcript of about 3 kb that encoded a sequence for the junction of human PG-M(V3) was detected in human brain and retina with probe C (Fig. 4, lane 3 for retina and lane 4 for brain).


DISCUSSION

On the basis of the present study and reported results(5, 6) , complete mouse and human cDNAs for PG-M(V3) were predicted to contain open reading frames of 1,965 and 1,968 nucleotides for proteins of M(r) 74,175 and M(r) 74,248, respectively. Northern blot analysis suggested that both mouse and human transcripts for PG-M(V3) had non-coding regions about 1 kb in size. The deduced amino acid sequences of both PG-M(V3) showed only the presence of a hyaluronan-binding domain sequence at the amino terminus and the two EGF-like repeat sequences, the C-type lectin-like sequence, and the CRP-like sequence at the carboxyl terminus(16, 17) . In addition, cDNA sequences of mouse and human PG-M(V3) revealed that both the amino-terminal and carboxyl-terminal portions showed completely identical sequences to those of mouse PG-M and human versican, respectively(5) . Together with Southern blot analysis data, this suggests that PG-M(V3) is generated from a single PG-M gene by alternative splicing.

PG-M(V3) essentially lacks the chondroitin sulfate attachment regions. We showed previously that PG-M had a strong inhibitory effect on the adhesion of cells to dishes precoated with various matrix proteins(8) . The active sites for the inhibitory activity of PG-M were chondroitin sulfate moieties(8) . Therefore, PG-M(V3) must be distinctly different in the activity from other forms of PG-M. However, in mouse and human PG-M(V3), there are two Ser-Gly and two Gly-Ser sequences that are presumed to be chondroitin sulfate attachment sites, although it is not clear whether chondroitin sulfate chains are actually linked to these sites. In addition, there are four potential N-glycosylation sites. Isolation of a molecule corresponding to PG-M(V3) is needed to examine whether PG-M(V3) is a proteoglycan or a glycoprotein.

Immunofluorescence staining and immunoblotting using monoclonal antibody to chick PG-M core protein suggested that core proteins of various sizes were detected in various tissues such as brain, aorta, and skeletal muscle(3, 18) . We have recently shown a tissue-dependent expression of alternatively spliced multiforms of PG-M(5) . In the present study, we showed that PG-M(V3) also existed in some tissues. PG-M(V3) could be detected in adult human cerebral cortex (brain) ( Fig. 4and Fig. 6), human retina (Fig. 4), adult human stomach (Fig. 6), fetal human liver (Fig. 6), and also in adult mouse brain (Fig. 2).

A number of recent studies have reported that chondroitin sulfate proteoglycans are found in nervous system tissues including the brain and retina(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) . Some have been shown to be developmentally regulated (20, 24) and involved in the regulation of neuronal patterning in the retina(29) . Moreover, versican has been shown to be expressed in human adult skin and is thought to be a regulator in this tissue(30) . PG-M(V3), because it has no chondroitin sulfate attachment region, may be a unique regulator that is required in these tissues.

In all PG-M (V0, V1, V2, and V3), the fact that there is always a link protein-like sequence at the amino-terminal end and EGF-like domains, a lectin-like domain, and a CRP-like domain at the carboxyl-terminal end suggests that these domains have minimum requirements for PG-M functions.

Hyaluronan must play an important role in the extracellular matrix of the brain, because there are several reports to suggest that hyaluronan-binding protein or hyaluronan-binding proteoglycan is found in the central nervous system (19, 31, 32, 33) and also in brain tumors (34) . It is especially noteworthy that brain-enriched hyaluronan-binding protein is only expressed in the brain(33) . Glial hyaluronan-binding protein (GHAP) is one of the hyaluronan-binding proteins in the extracellular matrix of the brain and is now thought to be a proteolytic product of versican and to correspond to its hyaluronan-binding amino-terminal domain(19, 35) . GHAP is mainly found in white matter, but it also isolated in gray matter(19, 35) . Our present results suggest a distribution and stage-dependent appearance of PG-M(V3) that is similar to that of GHAP, which further suggests that GHAP is also a proteolytical fragment of PG-M(V3) or is PG-M(V3) itself.

The COOH-terminal portion of PG-M has recently been shown to have carbohydrate binding activity to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner(10) . Moreover, these binding activities seem to need a whole set of EGF-, lectin-, and CRP-like domains(10) . Because not only all PG-M (V0, V1, V2, and V3) but also various aggregating proteoglycans, such as aggrecan(36, 37) , neurocan(20) , and brevican(38) , always contain this set of domains, the carboxyl-terminal portions of these proteoglycans might be needed to play a common functional role in carbohydrate binding activity. A possibility remains that other similar types of chondroitin sulfate proteoglycans, such as aggrecan(36, 37) , neurocan(20) , and brevican(38) , have PG-M(V3)-type mRNAs that have no chondroitin sulfate attachment region.


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 the 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) D32040 [GenBank]and D32039 [GenBank]for mouse and human PG-M(V3), respectively.

§
To whom correspondence should be addressed. Tel.: 052-264-4811 (ext. 2088); Fax: 0561-63-3532.

(^1)
The abbreviations used are: EGF, epidermal growth factor; CRP, complement regulatory protein; CS, chondroitin sulfate; GHAP, glial hyaluronan-binding protein; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s).


ACKNOWLEDGEMENTS

We are grateful to Drs. S. Nishida, N. Ogino, and M. Iwaki (Aichi Medical University) and Dr. Y. Honda (Kyoto University) for their continuous support and encouragement.


REFERENCES

  1. Kimata, K., Oike, Y., Tani, K., Shinomura, T., Yamagata, M., Uritani, M., and Suzuki, S. (1986) J. Biol. Chem. 261, 13517-13525 [Abstract/Free Full Text]
  2. Shinomura, T., Jensen, K. L., Yamagata, M., Kimata, K., and Solursh, M. (1990) Anat. Embryol. 181, 227-233 [Medline] [Order article via Infotrieve]
  3. Yamagata, M., Shinomura, T., and Kimata, K. (1993) Anat. Embryol. 187, 433-444 [Medline] [Order article via Infotrieve]
  4. Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469 [Abstract/Free Full Text]
  5. Ito, K., Shinomura, T., Zako, M., Ujita, M., and Kimata, K (1995) J. Biol. Chem. 270, 958-965 [Abstract/Free Full Text]
  6. Zimmermann, D. R., and Ruoslahti, E. (1989) EMBO J. 8, 2975-2981 [Abstract]
  7. Krusius, T., Gehlsen, K. R., and Ruoslahti, E. (1987) J. Biol. Chem. 262, 13120-13125 [Abstract/Free Full Text]
  8. Yamagata, M., Suzuki, S., Akiyama, S. K., Yamada, K. M., and Kimata, K. (1989) J. Biol. Chem. 264, 8012-8018 [Abstract/Free Full Text]
  9. LeBaron, R. G., Zimmermann, D. R., and Ruoslahti, E. (1992) J. Biol. Chem. 267, 10003-10010 [Abstract/Free Full Text]
  10. Ujita, M., Shinomura, T., Ito, K., Kitagawa, Y., and Kimata, K. J. Biol. Chem. 269, 27603-27609
  11. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  12. Shinomura, T., and Kimata, K. (1992) J. Biol. Chem. 267, 1265-1270 [Abstract/Free Full Text]
  13. Kusukawa, N., Uemori, T., Asada, K., and Kato, I. (1990) BioTechniques 9, 66-72 [Medline] [Order article via Infotrieve]
  14. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 [Medline] [Order article via Infotrieve]
  15. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5201-5205 [Abstract]
  16. Springer, T. A. (1990) Nature 346, 425-434 [CrossRef][Medline] [Order article via Infotrieve]
  17. Deak, F., Kiss, I., Sparks, K. J., Argraves, W. S., Hampikian, G., and Goetinck, P. F. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3766-3770 [Abstract]
  18. Yamagata, M., Saga, S., Kato, M., Bernfield, M., and Kimata, K. (1993) J. Cell Sci. 106, 55-65 [Abstract/Free Full Text]
  19. Bignami, A., Hosley, M., and Dahl, D. (1993) Anat. Embryol. 188, 419-433 [Medline] [Order article via Infotrieve]
  20. Rauch, U., Karthikeyan, L., Maurel, P., Margolis, R. U., and Margolis, R. K. (1992) J. Biol. Chem. 267, 19536-19547 [Abstract/Free Full Text]
  21. Iwata, M., and Carlson, S. S. (1993) J. Neurosci. 13, 195-207 [Abstract]
  22. Venstrom, K. A., and Reichardt, L. F. (1993) FASEB J. 7, 996-1003 [Abstract/Free Full Text]
  23. Perides, G., Rahemtulla, F., Lane, W. S., Ashert, R. A., and Bignami, A. (1992) J. Biol. Chem. 267, 23883-23887 [Abstract/Free Full Text]
  24. Maeda, N., Matsui, F., and Oohira, A. (1992) Dev. Biol. 151, 564-574 [Medline] [Order article via Infotrieve]
  25. Hennig, A. K., Mangoura, D., and Schwartz, N. B. (1993) Dev. Brain Res. 73, 261-272 [Medline] [Order article via Infotrieve]
  26. Hoffman, S., Crossin, K. L., and Edelman, G. M. (1988) J. Cell Biol. 106, 519-532 [Abstract]
  27. Perris, R., and Johansson, S. (1990) Dev. Biol. 137, 1-12 [Medline] [Order article via Infotrieve]
  28. Iijima, N., Oohira, A., Mori, T., Kitabatake, K., and Kohsaka, S. (1991) J. Neurochem. 56, 706-708 [Medline] [Order article via Infotrieve]
  29. Brittis, P. A., Canning, D. R., and Silver, J. (1992) Science 255, 733-736 [Medline] [Order article via Infotrieve]
  30. Zimmermann, D. R., Dours-Zimmermann, M. T., Schubert, M., and Bruckner-Tuderman, L. (1994) J. Cell Biol. 124, 817-825 [Abstract]
  31. Marret, S., Delpech, B., Delpech, A., Asou, H., Girard, N., Courel, M. N., Chauzy, C., Maingonnat, C., and Fessard, C. (1994) J. Neurochem. 62, 1285-1295 [Medline] [Order article via Infotrieve]
  32. Iwata, M., Wight, T. N., and Carlson, S. S. (1993) J. Biol. Chem. 268, 15061-15069 [Abstract/Free Full Text]
  33. Jaworski, D. M., Kelly, G. M., and Hockfield, S. (1994) J. Cell Biol. 125, 495-509 [Abstract]
  34. Delpech, B., Maingonnat, C., Girard, N., Chauzy, C., Maunoury, R., Olivier, A., Tayot, J., and Creissard, P. (1993) Eur. J. Cancer 7, 1012-1017
  35. Perides, G., Lane, W. S., Andrews, D., Dahl, D., and Bignami, A. (1989) J. Biol. Chem. 264, 5981-5987 [Abstract/Free Full Text]
  36. Doege, K., Sasaki, M., Horigan, E., Hassell, J. R., and Yamada, Y. (1987) J. Biol. Chem. 262, 17757-17767 [Abstract/Free Full Text]
  37. Baldwin, C. T., Reginato, A. M., and Prockop, D. J. (1989) J. Biol. Chem. 264, 15747-15750 [Abstract/Free Full Text]
  38. Yamada, H., Watanabe, K., Shimonaka, M., and Yamaguchi, Y. (1994) J. Biol. Chem. 269, 10119-10126 [Abstract/Free Full Text]

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