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The C-terminal domain V of the basement membrane proteoglycan perlecan was previously shown to play a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino-acid residues from Drosophila has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G-type (LG) and epidermal growth factor-like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yielded a 100-kDa protein which was folded into a linear array of three globular LG domains. Unlike the mouse counterpart, domain V from Drosophila was not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It showed moderate affinities for heparin and sulfatides but did not bind to chick α-dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V was also incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (≈ 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V. A strong expression was also found by in situ hybridization and immunohistology at various stages of embryonic development and expression was localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development.
Several proteins of the extracellular matrix have been shown to mediate cellular interactions which are instrumental for the survival and function of epithelial and other cells in both vertebrates and invertebrates . This is reflected in the sharing of important components involved in these processes across distant phyla. In Drosophila melanogaster such components include two laminin isoforms, collagen IV and integrin receptors [2–4]. Unique components include the cell-adhesive tiggrin and the proteoglycan papilin, and a major mammalian basement membrane proteoglycan, perlecan, has not yet been characterized in Drosophila. An analogue of perlecan has, however, been identified in Caenorhabditis elegans (unc-52) and differs to some extent in the modular structure of the protein core . This perlecan was previously shown to be important for the connection of muscles to the body wall.
Mammalian perlecan consists of a 480-kDa protein core which is substituted by three to four heparan sulfate or chondroitin sulfate chains [6,7]. It is produced during early and late stages of embryonic development in many organs and is localized in basement membranes, vessel walls, cartilage matrix and some other extracellular spaces . Perlecan-deficiency in mice caused multiple defects and early lethality [9,10]. Major phenotypes were defective basement membranes in the heart muscle and brain, exencephaly, an impaired endochondral ossification and an unstable cartilage matrix. The complex phenotype presumably reflects a diverse array of binding epitopes for several cytokines, laminins, nidogens, fibulins, heparin, sulfatides, α-dystroglycan and β1 integrins located in different domains of the protein core or on the heparan sulfate chains . These binding sites are particularly abundant in domains IV and V of perlecan.
Domain V is located at the C-terminal end of the protein core and consists of a tandem array of three laminin G-type (LG) modules and four epidermal growth factor-like (EG) modules. This domain can bind to the laminin-nidogen complex, fibulin-2, sulfatides, β1 integrins and α-dystroglycan [12–14]. It also binds weakly to heparin and can be modified by glycosaminoglycan substitution and endogenous proteolysis. Mapping with smaller fragments showed the involvement of EG or LG modules in these interactions and the requirement of two LG modules for the efficient binding of sulfatides and α-dystroglycan . Similar binding sites on two LG modules of the laminin α1 and α2 chains have been mapped by site-directed mutagenesis to several nonconsecutive basic residues [13,15]. The data also demonstrated a considerable or partial overlap of the binding epitopes for heparin, sulfatides and α-dystroglycan. The recent elucidation of the crystal structure of the laminin α2LG5 module has now allowed a spatial interpretation of these epitopes and the correct alignment and modelling of other LG modules .
The potential of the Drosophila model for genetic studies led us to search for its perlecan analogue. Based on two cDNA clones, it was feasible to determine the sequence of Drosophila perlecan domain V and to obtain it in recombinant form. This demonstrated a similar modular arrangement to that in mammalian perlecan but only a limited conservation of binding properties for carbohydrate and protein ligands derived from vertebrates. Specific antibodies could be generated and were useful for studying perlecan expression in cell cultures and tissues of Drosophila embryos.
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The sequence analysis clearly identified Drosophila perlecan domain V as an analogue of the same mouse perlecan structure, with 33% sequence identity and the module arrangement LG1EG1EG2LG2EG4LG3. The mouse EG3 module was replaced in Drosophila by a short proline-rich segment and a linking segment between EG4 and LG3 was also absent. This link contains a glycosaminoglycan attachment site, which is partially occupied in tissue-derived mouse and human perlecan, and its occupation was shown to inhibit cell adhesion and α-dystroglycan binding to domain V [12,14]. The complete sequence of C. elegans perlecan has recently been elucidated ; it demonstrated a similar modular arrangement for domain V as in Drosophila but had a low sequence identity (25%). This sequence also showed a replacement of the EG3 module by a 170-residue segment rich in threonine and glutamic acid and lacked the glycosaminoglycan attachment site of the link region.
A crystal structure has recently been established for the laminin α2LG5 module and showed a β-sandwich providing a hydrophobic core and various loops on the top and bottom of the core responsible for ligand binding . The study also demonstrated ligation of a single calcium ion and the importance of two aspartates in this ligation. These residues are conserved in many LG modules. It was furthermore speculated that calcium ligation plays an essential role in the interaction with the cellular receptor α-dystroglycan. An alignment of the mouse and Drosophila perlecan LG modules showed a good conservation of hydrophobic amino acids in the β strands, certain critical glycines and two cysteines involved in a disulfide bridge (Fig. 9). The conservation of calcium-ligating aspartates (or asparagines) was complete for mouse domain V but only existent for the LG1 module in the Drosophila domain V. This is of particular interest as α-dystroglycan binding to mouse perlecan domain V requires at least two LG modules  and depends on calcium-binding.
Figure 9. Multiple alignments of the LG1 to LG3 modules of mouse and Drosophila perlecan domain V and two selected mouse laminin LG modules from the α1 and α2 chain. The alignment is based on the crystal structure of α2LG5  indicating the position of 14 β strands (A–N), a single α-helix and two residues involved in a calcium ligation site (black spots). Conserved hydrophobic residues and glycines are highlighted in light grey and cysteines in dark grey. Thin single and double lines in the laminins identify basic stretches important for heparin, sulfatide and/or α-dystroglycan binding [15,16]. The positions of similar basic clusters in the perlecan sequences are underlined by thick lines.
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Recombinant Drosophila domain V was properly folded, as demonstrated by electron microscopy and a high content of β structure, which is typical for LG and EG modules. It was therefore a useful ligand to compare binding properties established for mouse perlecan domain V with the aim of selecting appropriate residues for site-directed mutagenesis. The studies were restricted to ligands obtained from vertebrate sources and showed a comparable binding of mouse and Drosophila perlecan domain V to heparin but not to several protein ligands. Binding of heparin to mouse domain V involved some of the five arginines in the EG2 module , but only two of them are conserved in Drosophila (Fig. 1). It is therefore possible that basic residues present in the H–I and J–K loops of the LG structure (Fig. 9), which are close in space and also contain critical residues for heparin binding to laminin α1LG4 and α2LG5 modules [13,15], are replacing the EG-binding epitope in Drosophila. The lack of binding of domain V to various mammalian basement membrane proteins, including laminin-1 and nidogen-1, indicates a divergent evolution of potential binding epitopes. Nevertheless, this information will now be useful for designing mutations in nonconserved loop regions (Fig. 9) in order to locate such binding epitopes on mouse perlecan domain V. Lack of conservation of binding epitopes seems, however, not to be always the case as mouse nidogen-1 binds to Drosophila laminin of the chain composition α3/α5β1γ1. This is explained by nearly identical binding epitopes being present in mouse and Drosophilaγ1 chains .
A further distinct binding could be demonstrated between Drosophila domain V and bovine brain sulfatides and was comparable to that reported previously for LG modules of mouse perlecan and laminin α1 and α2 chains [13–15]. Site-directed mutagenesis assigned the laminin binding epitopes to several basic residues in the F–G and H–I loops [13,15,16] and thus to regions which also have a basic character in perlecan domain V of Drosophila although not in that of mouse (Fig. 9). Here, therefore, we should again anticipate an evolutionary change in perlecan binding epitopes. Unlike mouse perlecan and laminins, Drosophila perlecan domain V did not bind to the dystroglycan receptor, which plays a major role in cell–matrix interactions of vertebrates . As discussed above, this could reflect the absence of two calcium-binding sites , or the divergence of dystroglycan receptors in invertebrates.
Drosophila laminin was previously shown to promote attachment, spreading and differentiation of various Drosophila cells mediated by integrins but failed to be a substrate for vertebrate cells [3,4,32]. Here we show that Drosophila perlecan domain V failed to bind two rat cell lines which are known to adhere to mouse perlecan and its domain V through β1 integrins [12,22]. The LG3 module of Drosophila domain V contains a potentially adhesive RGD sequence which is not conserved in mouse (Fig. 1 and Fig. 9). No binding to this RGD was observed in solid-phase assays with purified human αvβ3 integrin which binds avidly to such sequences of vitronectin and fibronectin. This could be explained by the location of the RGD in a short loop between β strands A and B of Drosophila (Fig. 9), a site which is not well accessible in the α2LG5 structure . Confirmation of this possibility will require studies with DrosophilaαPS2βPS integrin that binds readily to RGD sequences in the Drosophila substrates tiggrin, laminin α3/α5β1γ1 and ten-m [33,34].
The data with Drosophila cell cultures and tissue extracts demonstrated a perlecan protein core of at least 450 kDa, which can be substituted by heparan sulfate side chains. This large size agrees with genomic sequence data available from the Berkeley Drosophila Genome Project, which are still incomplete for the Ig modules. As for C. elegans perlecan , they demonstrate a good conservation of domains II–V, whereas domain I is not present and is apparently replaced by an extension of domain II with yet another 19 low-density-lipoprotein receptor type A modules and an Ig module (M. Schneider and S. Baumgartner, unpublished data). As domain I of vertebrates contains three major SGD attachment sequences for heparan sulfate [35,36] and, unlike in vertebrates , no additional substitution could be detected in the recombinant Drosophila domain V, it remains open as to which part of Drosophila perlecan is actually modified by heparan sulfate. A possible candidate is a single SGD sequence found in one IG module of domain IV. There is also another RGD sequence in Drosophila perlecan domain III which needs to be examined for cell adhesion activity.
At least two other heparan sulfate-containing proteins have been described so far in Drosophila, including a syndecan  and a glypican referred to as dally . Both proteins appear to be involved in growth factor signalling, and the heparan sulfate side chains were shown to be particularly important for proper signalling with different growth factors. Likewise, it could be envisaged that Drosophila perlecan, like its vertebrate analogues [39,40], will also interact with the fibroblast growth factor and its receptor signalling pathway, which appear to be conserved in evolution.
A second major aim of this work was to obtain suitable reagents for studying the expression of Drosophila perlecan during embryonic development.
There was an abundant expression of perlecan mRNA and protein in Drosophila embryos and its localization partially overlapped that of laminin α3/α5β1γ1, which can be found integrated in most basement membranes of the embryo. Mouse perlecan was shown previously to bind to laminins either by its heparan sulfate chains or through the protein core domain IV, the latter interaction being mediated by nidogen-1 [11–14,41,42]. It is likely that similar mechanisms exist in Drosophila, thus making the insect basement membranes comparable to those of vertebrates. Other basement membrane components or corresponding cellular receptors exist in Drosophila as well, such as dystroglycan (M. Schneider and S. Baumgartner, unpublished data), nidogen-1 (Drosophila Flybase) and BM-40 (E. Kohfeldt, S. Baumgartner and R. Timpl, unpublished data). At least dystroglycan shares the CNS and cardiac cell basement membrane staining with both laminin and perlecan (M. Schneider and S. Baumgartner, unpublished data). In this context, it is worth noting that the defects seen in perlecan-deficient mice [9,10], including neuronal ectopias, exencephaly and defective basement membranes ensheathing heart muscle, correlates well with the expression of Drosophila perlecan in brain and CNS and in cardiac cells, suggesting a conserved function of perlecan across distant phyla. This hypothesis can be tested once Drosophila perlecan mutants are available for analysis.