(Received for publication, August 31, 1995; and in revised form, January 23, 1996)
From the
The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
The most well characterized basement membrane proteoglycan is
perlecan, first isolated from the murine Engelbreth-Holm-Swarm (EHS) ()tumor(1) . This complex molecule is probably the
most abundant basement membrane proteoglycan, whose properties include
interaction with other basement membrane components to form the matrix,
participation in selective filtration, e.g. in the kidney
glomerulus, and interactions with cell surface integrin
receptors(2, 3, 4) . In addition, perlecan
may both sequester heparin-binding growth factors, such as fibroblast
growth factor 2, and potentiate their interaction with high affinity
receptors (5) . The entire sequences of murine and human
perlecan core proteins are now known and represent one of the largest
and most complex extracellular matrix protein structures
identified(6, 7, 8) . The core protein of M
396,000-467,000 consists of five
domains; the small N-terminal domain is the only one unique in
structure and potentially bears 1-3 heparan sulfate chains.
Domain II has homology to the low density lipoprotein receptor, while
domain III has homology to laminin 1 short arms. Domain IV consists of
14-21 Ig repeats most closely resembling those of neural cell
adhesion (N-CAM), and is potentially subject to alternate
splicing at the mRNA level(2) , while domain V contains
neurexin repeats and is homologous to the C-terminal domains of agrin,
and, to a lesser extent, the C-terminal globular region of laminin
chains(2, 3, 4) . Perlecan is widely
distributed in basement membranes and some other extracellular
matrices, as detected by monoclonal and polyclonal antibodies,
including some which are
domain-specific(2, 3, 4, 9, 10) .
The presence of other, distinct basement membrane proteoglycans is clear. Agrin is now recognized as a heparan sulfate proteoglycan (HSPG), and other HSPG core proteins may yet be characterized(11) . In addition, we have identified a chondroitin sulfate proteoglycan (CSPG), distributed in most, but not all, adult rat basement membranes (BM-CSPG; (12) and (13) ). It is immunologically unrelated to perlecan, is apparently regulated in embryonic development, and has been implicated in pathogenic changes in basement membranes accompanying several unrelated diseases(14, 15, 16) . All this may point to a role for BM-CSPG in basement membrane stability.
The EHS tumor, while containing perlecan as its major proteoglycan component, has not been investigated for its galactosaminoglycan-bearing proteoglycans. Perlecan itself probably represents <10% of the basement membrane components present in the tumor matrix. Chondroitin sulfate has been detected as a minor proportion of the glycosaminoglycan, representing from <5% to 19% of the tumor matrix glycosaminoglycan content in various preparations (17, 18, 19) . The core proteins bearing galactosaminoglycan have not been determined. One high density, small proteoglycan bearing both chondroitin and heparan sulfate chains has been identified(21) , but is a poor antigen, and so its relationship, if any, to known species such as syndecans (2) or a fragment of perlecan is unclear. In this work, we utilize immunological and molecular techniques to characterize two chondroitin/dermatan proteoglycans (CS/DSPGs) of the EHS tumor. One is perlecan itself, which is present as both a heparan sulfate-substituted species and as a hybrid heparan/dermatan sulfate proteoglycan. The second is BM-CSPG, present as a CSPG and confirmed to be distinct from other proteoglycans.
The
concentrate was chromatographed on a column of DEAE-Sephacel (2.5
40 cm; Pharmacia Biotech Inc.) equilibrated with 6 M urea, 0.05 M Tris-HCl, pH 6.8. After sample loading, the
column was washed with 5 bed volumes of the equilibration buffer.
Subsequently, the column was eluted with 3 bed volumes each of 0.3 M NaCl and 2 M NaCl in 6 M urea, 0.05 M Tris-HCl, pH 6.8. The 2 M NaCl eluate was dialyzed
against 6 M urea, 0.05 M Tris-HCl, pH 6.8, and
chromatographed on a DEAE-Sephadex column (10
40 cm)
equilibrated with 6 M urea, 0.05 M Tris-HCl, pH 6.8.
The column was washed with 5-7 bed volumes of the equilibration
buffer followed by elution with a linear gradient of 0.1 M NaCl to 2 M NaCl in 6 M urea, 0.05 M Tris-HCl, pH 6.8. Approximately 3-ml samples were collected and
monitored for uronic acid(22) . Fractions containing uronic
acid were pooled, dialyzed against distilled H
O (without
protease inhibitors), and lyophilized.
The lyophilized material was
resolubilized (5 mg/ml) in 4 M guanidinium HCl, 0.05 M Tris-HCl, pH 7.4 (with protease inhibitors) and chromatographed on
a column of Sepharose CL-4B (2.5 100 cm; Pharmacia).
Approximately 5-ml fractions were collected and analyzed for uronic
acid. Fractions containing uronic acid were appropriately pooled,
dialyzed against distilled H
O, lyophilized, and stored
desiccated at 4 °C for further analysis. The column was calibrated
with rooster comb hyaluronan for the V
and
[
H]proline for the V
.
The column flow-through, including a column volume of PBS wash, was collected, and 100 µl of R63 antiserum was added before incubation on a rotator for 90 min at 37 °C. Three mg of Protein A-Sepharose (Pharmacia) was preincubated with 10% fetal bovine serum in PBS containing 0.02% sodium azide to block nonspecific binding sites, washed copiously by centrifugation and resuspension in PBS, then mixed with the proteoglycan/antibody preparation for 45 min at 37 °C. The immunoprecipitates were washed repeatedly with PBS. Immunoprecipitated proteoglycans were eluted with two 0.5-ml aliquots of 100 mM glycine-HCl, pH 2.8. An equal volume of 0.2 M NaCl, 50 mM sodium acetate, pH 5.5, was added, and the pH was adjusted, if necessary, to 4.0. Aliquots of 200 µl/well were applied to DEAE-membrane (NA45; Schleicher & Scheull, Keene, NH) assembled in a Bio-Rad slot blot apparatus, for 2 h at room temperature. Under these buffer conditions, the immunoprecipitated proteoglycans, but not the majority of the R63, bound to the membrane.
The membrane was washed with 0.2 M NaCl, 50 mM sodium acetate, pH 4.0, then blocked with 0.5% dried milk, 1% normal goat serum in PBS containing 0.05% Tween 20 (PBST). After brief washing in PBST, duplicate wells were incubated with 1:1000 dilutions in PBST of preimmune R63, immune R63 (negative and positive controls respectively), or 1:50 dilutions of 5A3 mouse monoclonal antibody against BM-CSPG(12) , an irrelevant monoclonal antibody AY8 of the same IgG subclass, and 11B4 monoclonal antibody against rat perlecan core protein. Additional wells received no primary antibody. After a 1-h incubation at 37 °C, the membrane was washed extensively with PBST, followed by 1:2000 dilutions in PBST of alkaline phosphatase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG. Additional washes in PBST after incubation for 1 h at 37 °C with secondary antibodies was followed by color development, according to the manufacturer's protocol (Bio-Rad). The blots were photographed immediately. The entire experiment was repeated three times, with identical results.
Positive plaques were picked and subjected to additional rounds of screening and purification before obtaining Bluescript SK(-) plasmids by in vivo excision. DNA sequencing was performed in both directions by modified dideoxynucleotide chain termination methods, using Sequenase 2.0 (U. S. Biochemical Corp.), using SK and T7 primers.
For fusion
protein production, SLOR Escherichia coli bacteria containing
four clones (4a, 5a, 11a, and 15a) in Bluescript SK(-) were grown
to midlog phase at 37 °C, and then
isopropyl-1-thio--D-galactopyranoside was added to 10
mM final concentration. The cells were further incubated until
reaching stationary phase. After centrifugation at 1600
g for 10 min, the bacterial pellets were harvested and resuspended
in 10 mM Tris, 150 mM NaCl, pH 7.4, containing 1% SDS
and sonicated for 1 min. Samples were solubilized in reducing SDS-PAGE
loading buffer, and proteins were resolved on 3-15%
polyacrylamide gels. Electrophoretic transfer to nitrocellulose was
followed by blocking in 1% fat-free milk in Tris-buffered saline as
before (9) and probed with 1:1000 dilutions of polyclonal
antibodies R63 and EY#S for 2 h at room temperature. After washing, the
membranes were incubated in a 1:2000 dilution of alkaline
phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) for 1 h at room
temperature, and color was developed with the Bio-Rad reagent,
following the manufacturer's procedure.
Figure 1: Gel chromatography on a column of Sepharose CL-4B of purified EHS tumor proteoglycans. Three pools of uronic acid-containing fractions were collected for further analysis as shown.
Figure 2: Immunoblotting of pool 1 proteoglycans with R36, a polyclonal antibody recognizing chondroitin/dermatan sulfate stubs remaining on core proteins after chondroitinase ABC treatment. Lanes 1 and 7 are standards whose molecular mass in kilodaltons is indicated. Lane 2, untreated proteoglycan; lane 3, proteoglycans treated with chondroitinase ABC and heparinases II and III; lane 4, proteoglycans treated with chondroitinase ABC only; lane 5 contains heparinases II and III only, while lane 6 contains chondroitinase ABC alone.
Since the
larger species had a core protein of approximate M
400,000, an obvious candidate molecule was perlecan.
Therefore, five core protein-specific monoclonal antibodies recognizing
murine perlecan were used separately in Western blotting of P1 and P2
material following treatment with chondroitinase ABC and/or heparinase
III. These all recognized polypeptides of an identical high molecular
mass, the largest of which was of M
400,000 (Fig. 3), confirming that the high molecular weight core protein
was perlecan. The perlecan core protein could be resolved after
heparinase III treatment alone and was therefore also present as a
proteoglycan species bearing only heparan sulfate chains (lane
2, Fig. 3, A, B, and C).
Perlecan in the form of an HSPG has been reported
previously(1, 17, 18, 19) . These
five antibodies recognized at least four different epitopes, and three
have been mapped to epitopes within domains III, IV, or V of the core
protein(9) . Two different polyclonal antibodies were also
tested, one raised against perlecan, the other against an HSPG prepared
from the PYS-2 murine endodermal cell line. Both gave results virtually
identical with the five monoclonal antibodies (not shown). In addition,
three other features emerged. First, in the P2 material, the monoclonal
antibodies were able to detect small amounts of M
400,000 polypeptide in samples not enzyme-treated (lane
1, Fig. 3B) or treated with chondroitinase ABC
alone (lane 4, Fig. 3B). This shows that some
perlecan can be purified, which either lacks or has extremely small
glycosaminoglycan chains, in addition to some which contains only
chondroitin/dermatan sulfate chains. Second, after heparinase III
treatment, at least two perlecan core proteins were resolved as a
closely spaced doublet, whereas only a single core was detected after
chondroitinase ABC treatment. The latter corresponded to the largest
form seen after heparinase III treatment (lanes 2, Fig. 3). The species with higher mobility was not, however, a
protein contaminant from the heparinase III, since no polypeptides were
detected by any antibody in lanes where heparinase III alone had been
loaded (lanes 5, Fig. 3). Third, no perlecan antibody,
either monoclonal or polyclonal, recognized the CS/DSPG with a core of M
200,000.
Figure 3: Immunoblotting of pool 2 proteoglycans with monoclonal antibodies G9L1 (A), C11L1 (B), and A10L4 (C) against perlecan core protein domains III, IV, and V, respectively. In each blot, the proteoglycans are untreated (lanes 1), heparinase III-pretreated (lanes 2), heparinase III- and chondroitinase ABC-pretreated (lanes 3), or chondroitinase ABC-pretreated (lanes 4). Lanes 5 contain heparinase III only, while lanes 6 contain chondroitinase ABC only. An arrow marks the location of a molecular mass standard (in kilodaltons).
Figure 4:
Immunoblotting of pool 2 proteoglycans
with R63 polyclonal antibody against EHS tumor matrix
chondroitin/dermatan sulfate proteoglycans. The proteoglycans are
untreated (lane 1), heparinase III-pretreated (lane
2), heparinase III- and chondroitinase ABC-pretreated (lane
3), or chondroitinase ABC-pretreated (lane 4). The M
200,000 CS/DSPG core protein is indicated
by arrowheads in lanes 3 and 4. The arrows denote the migration of molecular mass standards in
kilodaltons.
Figure 5: Indirect immunofluorescence microscopy of human skin (a and b) and rat kidney cortex (c) stained with R63 antibody against EHS tumor CS/DSPGs. Basement membranes of the dermal-epidermal junction (J) and papillary dermal capillaries (arrow, a), sweat glands and ducts (S) and their associated vasculature (V in b), and kidney tubules (T), Bowman's capsule (P), and glomerular mesangium (M in c) are stained. Glomerular capillary loop basement membranes are virtually unstained. Bar = 100 µm.
Figure 6: L2 cell culture medium proteoglycans immunoblotted with R63 against EHS tumor CS/DSPGs (A), 11B4 monoclonal antibody against rat perlecan core protein (B), and R36 against chondroitin/dermatan sulfate chain stubs remaining attached to core proteins after chondroitinase ABC treatment (C). In each case, lanes 1 contain untreated proteoglycans, in lanes 2 the proteoglycans are heparinase III-treated, in lanes 3 the samples are heparinase III- and chondroitinase ABC-treated, and in lanes 4 chondroitinase-treated only. The BM-CSPG core proteins are labeled with arrowheads, and the positions of molecular mass standards, in kilodaltons, are shown by arrows.
The antiserum R63 was used to screen an expression
cDNA library from L2 cell mRNA in the Uni-ZAP vector. Of 11 clones
detected from preliminary screening and subsequent rounds of clonal
selection, two different polypeptides were identified by DNA
sequencing. Two overlapping clones of 2.2 kb yielded portions of domain
IV of perlecan core protein, as judged by very high homology to the
previously reported murine and human sequences. These were only weakly
detected by the R63 serum. The remaining nine clones, all overlapping,
gave a predicted amino acid sequence not related to any lodged with
EMBL or GenBank. In total these latter clones encompassed 3.4kB,
including an open reading frame of 2.9 kb and 0.5 kb of 3`-noncoding
sequence. This sequence will be reported elsewhere. The two bacterial
clones expressing a portion of rat perlecan core protein (5a and 11a),
and two of the nine putatively expressing BM-CSPG (4a and 15a) were
grown in liquid culture in the presence of
isopropyl-1-thio--D-galactopyranoside to induce fusion
protein expression. Bacterial lysates were probed by Western blotting
with R63 or EY#S, a polyclonal anti-murine perlecan serum (Fig. 7). While clone 5a and 11a fusion proteins could be
detected with anti-perlecan sera, as expected, clone 4a and 15a fusion
proteins could not. This was consistent with these latter proteins
being a portion of a distinct gene product. Clone 4a and 15a fusion
proteins were strongly detected by R63, however, while 5a and 11a
perlecan clones were only weakly detected. As predicted from
immunological studies, therefore, at least two distinct basement
membrane proteoglycan core proteins are present within the EHS tumor
matrix.
Figure 7: Immunoblots of bacterial lysates expressing fusion proteins of four distinct clones derived from antibody screening of a L2 cell cDNA expression library, probed with R63 (A) and EY#S polyclonal antibody against perlecan (B). Clones 5a and 11a correspond to portions of rat perlecan core protein, while 4a and 15a are putatively BM-CSPG core protein. Arrowheads mark the specific polypeptides detected by the antibodies. Fusion protein 5a is expressed at low levels and is not clearly visible in A. Molecular mass standard migration is indicated (in kilodaltons).
Figure 8: Slot blot of R63-immunoprecipitated proteoglycans probed with preimmune R63 (1 and 2), immune R63 as a positive control (3 and 4), 5A3 monoclonal antibody against BM-CSPG (5 and 6), irrelevant monoclonal antibody AY8 (7 and 8), 11B4 monoclonal antibody against rat perlecan core protein (9 and 10), and alkaline phosphatase-conjugated goat anti-rabbit IgG (11) or goat anti-mouse IgG (12) only.
Figure 9: Immunoblot, with R63, of pool 2 proteoglycans from the EHS tumor matrix, untreated (lane 2), heparinase III-treated (lane 3), heparinase III- and chondroitinase ABC-treated (lane 4), or heparinase III- and chondroitinase ACII-treated (lane 6). Chondroitinase ABC only is shown in lane 5 or chondroitinase ACII only in lane 7. Lane 1 shows molecular mass standards in kilodaltons. BM-CSPG core protein is marked with arrowheads.
Many basement membrane components have been purified and characterized from the EHS tumor matrix, and, in some cases, their first isolation from this source has been the catalyst for new directions of research. Perlecan is such a case and remains the best characterized basement membrane proteoglycan. It is now clear, however, that not only is the extracellular matrix from the tumor very complex, containing a number of macromolecules and growth factors(1, 17, 18, 19, 30, 31) , but the spectrum of basement membrane macromolecules described from this source is only a subset of those found in mammalian basement membranes in vivo. For example, the laminin family has expanded from the prototypical heterotrimer, first isolated from the EHS tumor, and it is now appreciated that at least 10 distinct chains can be identified from a variety of sources(32) .
While perlecan is the most abundant proteoglycan in the EHS tumor matrix, other smaller proteoglycans have previously been described. A small high density HSPG has been purified, apparently bearing shorter glycosaminoglycan chains than the lower buoyant density perlecan. This may be a fragment of perlecan, but it has also been suggested that it has only a weak immunological relationship to the larger, low density species(33) . It seems quite likely that while perlecan may be cleaved to generate smaller proteoglycans, there are additional unrelated HSPGs, with distinct core proteins. One small, high density hybrid proteoglycan has been described which bears both chondroitin and heparan sulfate chains(21) , and it has been postulated to be related to cell surface syndecan, possibly syndecan 1(2) . However, direct immunological data are currently lacking, preventing a firm identification of this molecule. Previous analyses indicate that up to 20% of the tumor matrix glycosaminoglycans are chondroitin or dermatan sulfates, but no core proteins have been associated with these chains (17, 18, 19) .
This report identifies two distinct proteoglycans as galactosaminoglycan-bearing. The first is perlecan itself, which, in addition to being substituted with heparan sulfate alone, was also present as a hybrid, with additional chondroitin sulfate chains. Indeed, small amounts of perlecan bearing galactosaminoglycan in the absence of heparan sulfate could also be detected, as well as very small amounts bearing no, or very small, glycosaminoglycans. This data are consistent with other observations. First, a cell line derived from the EHS tumor (34) was shown to synthesize perlecan as a hybrid molecule, and a similar form with heparan and dermatan sulfate chains was purified from human placenta (35) and bovine cartilage(36) . This indicates that perlecan can occur as a hybrid in vivo and, therefore, is one member of the overall basement membrane CS/DSPGs. One report also shows that perlecan can be found in vivo as a CS/DSPG, with no heparan sulfate chains(37) . However, it has not been reported previously that perlecan may exist in the EHS tumor matrix in these various forms. The impact of alternate glycosylation of the perlecan core protein is not understood, but, in the light of the potential importance of heparan sulfate in its interactions with integrins and growth factors(5, 38) , it is possible that substitution with dermatan sulfate chains in place of, or in addition to, heparan sulfate may impact the biological activity of perlecan. Further, perlecan forms bearing heparan sulfate yielded two core proteins, unlike the single species resolved after chondroitinase ABC treatment. The reasons for this are unclear, but it probably does not result from proteolytic activity in the heparinase III enzyme. Not only were protease inhibitors present, but perlecan from other sources (L2 cells, human keratinocytes, or murine PYS-2 or PFHR-9 cells) can be resolved as single core proteins after identical enzyme treatments. It is possible that endogenous proteolytic activity in the tumor matrix is responsible, but why heparan sulfate, but not galactosaminoglycan-substituted forms, should be more susceptible is unclear, although potentially interesting. It is currently unknown where in the core protein perlecan is substituted with chondroitin sulfate and in which tissues this occurs. Indeed, the site of heparan sulfate chain substitution is inferred from the SGD sequences in domain I, but has yet to be confirmed directly(2) .
The other proteoglycan identified was immunologically related to BM-CSPG and probably is identical with this for the following reasons. First, the glycosaminoglycan and core protein characteristics are very similar, and, since R63 stained only basement membranes, the proteoglycan is basement membrane-restricted in its distribution. Staining of adult rat kidney sections showed marked depletion in the glomerular capillary loop basement membrane, entirely consistent with our previous reports (12, 13) for BM-CSPG. Immunopurification of L2 cell proteoglycans by R63, followed by slot-blotting with previously characterized monoclonal antibodies, confirmed the presence of both BM-CSPG and perlecan reactivity in the antiserum raised against EHS tumor matrix CS/DSPGs. Screening of an expression cDNA library with the R63 antibody also led to the identification of two distinct proteins. One was perlecan, on the basis of high homology of the (rat) sequence to mouse and human perlecan domain IV. The other was unique and is currently being completed. However, preliminary data indicate an mRNA of approximately 4.2 kb, consistent with a protein smaller than perlecan (12-14 kb), and a sequence with total lack of identity or homology with perlecan or other basement membrane components. The sequence indicates a five domain structure in a head-rod-tail configuration, with large domains predicted to form coiled-coil structures(39, 40) . There is no structural similarity with perlecan or agrin, therefore, nor with any currently reported chondroitin/dermatan sulfate proteoglycan. Since at least two distinct proteoglycans in basement membranes bear galactosaminoglycans, it is appropriate to name these precisely to avoid confusion. As we have reported in preliminary communications, BM-CSPG will be called bamacan (basement membrane-associated chondroitin sulfate proteoglycan).
The
presence of BM-CSPG in the EHS tumor matrix has not been noted before,
but is consistent with other features of this matrix. We did not detect
the presence of a small hybrid proteoglycan that was noted
previously(21) , even though we used antibodies capable of
detecting all CS/DSPGs (R36) by virtue of its reactivity to the
unsaturated terminal uronic acid residues created by chondroitinase
treatments. Nor did we detect small HSPGs in these proteoglycan
preparations, but lack of appropriate immunological reagents may
explain this, assuming that the core proteins are unrelated to
perlecan. One such HSPG with a core protein of M
40,000 has been reported from liver tissue(41) , for
example. Therefore, while we have determined the presence of two
distinct proteoglycans in the EHS tumor matrix, BM-CSPG being a minor
component consistent with earlier findings, we do not rule out the
possibility of further basement membrane proteoglycans in this matrix.
It would not be surprising, given the large number of type IV collagen
chains and laminin isoforms now recognized, that multiple species of
proteoglycan are also present in tissue basement membranes, perhaps
regulated in developmental and/or tissue specific patterns. Our
information on BM-CSPG indicates that it is developmentally regulated,
but also affected in diseases such as diabetes and polycystic kidney
disease(14, 15) . We have also gained indications that
basement membrane chondroitin sulfate may be substantially missing in
the dermal-epidermal junction of patients with dystrophic forms of
epidermolysis bullosa(42) . Perlecan core protein was present
in an apparently normal pattern, but since this may be a hybrid
proteoglycan, the possibility arises that its glycosylation may be
affected, in addition to any effects on BM-CSPG, in disease states.