(Received for publication, October 14, 1994; and in revised form, November 11, 1994)
From the
Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5` end as well as in frame a stop codon at the 3` end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.
Perlecan, a heparan sulfate proteoglycan with a large (M = 400,000) core protein, is a major
component of all basement membranes. The primary structure deduced from
sequencing murine and human cDNA clones indicates that the core protein
has five distinct domains (Noonan et al., 1988, 1991; Murdoch et al., 1992; Kallunki and Tryggvason, 1992). The N-terminal
end of the core protein, domain I, consists of amino acid sequences
unique to perlecan and has three potential heparan sulfate attachment
sites. Domain II is homologous to low density lipoprotein binding
region of the low density lipoprotein receptor. Domain III consists of
three cysteine-free laminin-like globules that alternate with
cysteine-rich EGF(
)-like units and is most homologous to the
N-terminal third of laminin A chain. Domain IV contains variably
spliced immunoglobulin type repeats similar to those found in neural
cell adhesion molecule. Domain V, at the C terminus of perlecan,
consists of three cysteine-free neurexin-like globules (Ushkaryov et al., 1992) that alternate with EGF-like units and is most
homologous to the C-terminal third of agrin (Patthy and Nikolics,
1994).
Perlecan is implicated in multiple and diverse functions
(Timpl, 1993; Hassell et al., 1993). Determining the functions
of the specific structural domains is critical to understand the
functional diversity of this complex molecule. The heparan sulfate
chains affect filtration of macromolecules (Farquhar, 1981), while the
core protein interacts with itself (Yurchenco et al., 1987)
and other basement membrane components, such as fibronectin (Hermans et al., 1990) and entactin/nidogen (Battaglia et al.,
1992). A majority of the core protein interactions with other matrix
components contribute to the assembly (Laurie et al., 1986) or
maintenance of the matrix ultrastructure (Laurie, 1985). Both the core
protein and the heparan sulfate chains can also bind growth factors and
affect hemostasis (Timpl, 1993). Several studies have shown that cells
can attach to the core protein; however, the signaling pathways
involved are not yet understood. Hayashi et al. (1992)
demonstrated attachment of endothelial cells to the intact perlecan
core protein, and this attachment could be partially inhibited by the
RGD synthetic peptide as well as an anti-1 integrin antibody.
Murine perlecan has a single RGD sequence located in the second
globular subdomain of domain III (Noonan et al., 1991). In the
current study, we prepared a cDNA construct for domain III of perlecan
from existing cDNA clones and used this construct in a mammalian
expression vector system to express a recombinant domain III of
perlecan. We show that the recombinant protein is secreted, folded
properly, and is biologically active in supporting cell adhesion
through cellular interactions of its RGDS sequence.
Figure 1:
Northern analysis of four clonal HT1080
cell lines permanently transfected with domain III mRNA. The
recombinant produce is a 3.6-kb mRNA. Native perlecan mRNA
(12-13 kb) is not detected.
Synthesis and secretion of the recombinant protein into
the medium was analyzed in [S]methionine-labeled
clonal cell line 6 by immunoprecipitation with a polyclonal
anti-perlecan antiserum and followed by SDS-PAGE under reducing
conditions (Fig. 2). After 20 min of pulse-labeling with
[
S]methionine, a 160-kDa recombinant protein
could be immunoprecipitated from the cell lysate extracts. A similarly
sized recombinant protein could be immunoprecipitated from the medium
after a chase period of 5 h with unlabeled methionine-containing
medium. Thus, the domain III cDNA construct is transcribed, translated,
processed, and secreted as a protein that migrated with an apparent
molecular mass of 160 kDa under reducing conditions.
Figure 2:
Synthesis and secretion of domain III in
transfected HT1080 clonal cell line. Cells were metabolically
radiolabeled with [S]methionine, pulsed for 20
min, and chased for 5 h with unlabeled methionine. Recombinant domain
III was immunoprecipitated from cell layer extracts and media with
antisera to perlecan. The samples were reduced with dithiothreitol and
analyzed by SDS-PAGE. CL, extracted cell layer; M,
medium.
Figure 3: Western blot of purified domain III. Purified domain III (not reduced) was resolved in a 5% SDS-polyacrylamide slab gel and transferred by Western blotting to an Immobilon filter. Domain III was detected by a polyclonal anti-perlecan antiserum and secondary anti-rabbit goat IgG conjugated to horseradish peroxidase.
Figure 4: Digestion of domain III with V8 protease (at a ratio of 100:1) for 0-16 h, as indicated. Digested samples (not reduced) were resolved by SDS-PAGE on a 5% polyacrylamide slab gel and the fragments viewed by Coomassie Blue staining.
Examination of domain III in the electron microscope (Fig. 5) revealed a rod-shaped structure with several globules. This is consistent with structural predictions from sequence data and sequence homology with laminin short arms (Noonan et al., 1991). Domain III lengths measured 20 ± 1 nm long (n = 48). This length is approximately 25% of the total core protein length (Laurie et al., 1988; Paulsson et al., 1987) and this compares favorably with molecular mass of domain III (126 kDa) relative to the total mass (396 kDa) of perlecan core protein.
Figure 5: Electron micrographs of rotary shadowed domain III. a, low power field. The bar represents 15 nm. b-g, high power micrographs of domain III in which the globules are more apparent. The bar represents 10 nm. h, schematic diagram of domain III.
Figure 6:
Dose-dependent adhesion of mouse mammary
tumor cells to domain III and laminin.
[H]Thymidine-labeled MMT 060562 cells were added
to 24-well tissue culture dishes (5
10
cells/well)
coated with domain III or laminin as indicated. Attachment is expressed
as a percent of total cells added. Mean basal level of attachment,
defined as cell attachment to bovine serum albumin-coated wells, was
subtracted from each observation. Each data point is an average of two
independent observations.
To determine if the cell adhesive activity of domain III is due to cellular recognition of the RGDS sequence, attachment to domain III was assessed in the presence of increasing amounts of soluble RGDS, added to the medium at the time of plating the cells (Fig. 7). As little as 10 µg/ml RGDS inhibited cell attachment completely, while the control GRADSP peptide had very little effect.
Figure 7: RGDS mediated inhibition of cell attachment to domain III. Cells, preincubated with increasing amounts of RGDS or GRADSP, were allowed to attach to wells coated with 1 µg of domain III. Percent attachment was calculated as in Fig. 6, and each data point is an average of two observations.
To determine whether the RGDS-mediated attachment activity of domain III, demonstrated above, is present in intact native perlecan and, hence, biologically significant, attachment inhibition assays with intact perlecan were also performed (Fig. 8). MMT cells were incubated in domain III (1 µg/well)-coated wells in the presence of 100 µg/well soluble perlecan or laminin, as a control. Intact perlecan completely inhibited attachment to domain III, while laminin had no effect.
Figure 8: Intact perlecan, but not intact laminin (100 µg/well), inhibits cell adhesion to domain III. The results represent the mean of three measurements ± S.D.
Perlecan is known to support attachment of various types of cells (Clement and Yamada, 1990; Clement et al., 1989). More recently, murine perlecan has been shown to support adhesion of aortic endothelial cells involving integrins binding an RGD sequence (Hayashi et al., 1992). Domain III of perlecan contains the single RGD sequence present in murine perlecan. Since it is difficult to purify large quantities of perlecan and further purify proteolytically cleaved domains, we produced domain III as a recombinant protein and evaluated its cell attachment activity.
HT1080 cells transfected with the domain III construct, produced a 4-kb domain III transcript, as evidenced by Northern analysis on total RNA. The 4-kb message was translated into a 130-kDa secreted recombinant protein, whose apparent molecular mass increased to 160 kDa upon reduction. The 130-kDa size obtained for unreduced domain III on SDS-PAGE is close to the 126-kDa size predicted from its molecular mass. The increase in apparent molecular mass to 160 kDa upon reduction is due to the unfolding in its cysteine-rich EGF-like units. Even though recombinant domain III was derived from the core protein of a proteoglycan, the recombinant protein produced did not contain heparan sulfate side chains. The side chains are thought to be attached to domain I of perlecan, and although the first 14 amino acids of domain I are part of this construct, it does not contain the heparan sulfate attachment signal.
V8 protease digests of domain III release the same size 44- and 46-kDa fragments that have been previously shown to be derived from the EGF-rich regions in domain III of native perlecan (Ledbetter et al., 1987; Noonan et al., 1991). This indicates that the recombinant domain III is folded similarly, if not identically, to that domain in native perlecan. Interestingly, although the total number of cysteines in domain III is an even number, each of the four EGF subdomains in domain III contain an odd number of cysteines (Noonan et al., 1991). This suggests that the cysteine-cysteine pairing goes across the cysteine-free globules in domain III. Furthermore, in rotary shadow electron micrographs, the alternating globular units and cysteine-rich EGF units of recombinant domain III have a beaded appearance as in intact perlecan (Paulsson et al., 1987; Laurie et al., 1988). These observations suggest that the primary and secondary structure of recombinant domain III is similar, if not identical, to domain III of native perlecan. The recombinant protein supported attachment in a dose-dependent manner with 40% of the cells attached at the maximum dose. All of this attachment, however, could be abolished with the RGDS synthetic peptide. This indicates that the RGDS sequence is the only cell binding site in the recombinant protein. Hayashi et al.(1992) reported a considerably higher percent attachment of cells to the intact core protein, but this attachment could be reduced by only 40% in the presence of RGDS peptide. Taken together, these findings suggest that the RGDS-dependent site is in domain III and that the RGDS-independent cell adhesion sites in perlecan are in the other domains. To address the possibility that subtle differences in the folding of domain III is exposing a RGDS site which may be cryptic in native perlecan, we examined the ability of intact perlecan to inhibit cell attachment to recombinant domain III. Indeed, intact perlecan, but not laminin, could compete with domain III for cell surface receptors and block attachment to domain III. Although mouse laminin also supports integrin-mediated cell attachment, its RGDS sequence in the P1 region is cryptic and can be unmasked only by proteolysis (Aumailley et al., 1990). Most laminin-mediated adhesion is thought to occur through the E8 region (Deutzmann et al., 1990).
The role of extracellular matrix molecules in
controlling cell differentiation and gene expression is well known.
Cell surface integrin receptors, considered to be the major players in
regulating these processes, affect cellular signaling pathways, such
as, tyrosine phosphorylation, cytoplasmic alkalization, and
intracellular Ca fluctuations (reviewed by Juliano
and Haskill(1993)). In addition, integrin-mediated attachment of cells
to the extracellular matrix may be essential for the survival of
anchorage dependent cells (Meredith et al., 1993). Our study
suggests that perlecan may be an extracellular matrix ligand in these
phenomena.
The RGDS sequence in domain III of murine perlecan is also found in Caenorhabditis elegans (Rogalski et al., 1993). Therefore, this RGDS site is conserved in organisms from C. elegans to mouse, and perlecan presumably serves as a major integrin ligand in organisms up to rodents in the evolutionary hierarchy. The loss of this RGDS sequence in human perlecan (Kallunki and Tryggvason, 1992; Murdoch et al., 1992) raises the possibility of ligand degeneracy and the same cell signaling pathway being adequately induced by some other RGDS-containing ECM component.