(Received for publication, September 23, 1994; and in revised form, November 30, 1994)
From the
We showed previously that the alternative splicing of
chondroitin sulfate attachment domains (CS and CS
) 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.
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())-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
and CS
).
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.
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.
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 [
-
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
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
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.
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.
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) .
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.
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).
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 74,175 and M
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.
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.