Perlecan, the major HS PG of basement membranes and other connective tissues, is a modular molecule with five structural domains. The molecular mass of the protein core is ~ 470 kDa whereas the molecular mass of the PG that includes the covalently attached GAG chains is in the range 600–700 kDa. The complete PG has been expressed in both HEK-293 and JAR choriocarcinoma cells and shown to interact in an HS-dependent fashion with activin A, a member of the TGF-β superfamily . Of the five domains, the N-terminal domain I is the smallest with a molecular mass of ~ 20 kDa, and has been shown to contain a sperm protein, enterokinase and agrin module similar to that seen in mucins, and three well-characterized GAG-attachment sites, identified by the sequence Ser–Gly–Asp at residues Ser65, Ser71 and Ser76. Amino acid residues upstream of this region, as well as domains to the C-terminus, have been shown to be important in determining what type of GAG ultimately decorates each of the serine residues . Domain I was first expressed in CHO cells , then in insect cells  and has subsequently been expressed in COS-7  and HEK-293 cells [123-127]. In all of the mammalian systems, recombinant domain I was expressed as a PG mostly decorated with HS, whereas expression in conjunction with C-terminal domains of perlecan or as a fusion protein with enhanced green fluorescent protein resulted in only HS decoration [120, 124]. The HS attached to domain I was shown to be important for the binding of growth factors such as fibroblast growth factor (FGF)-2 [8, 125, 128], vascular endothelial growth factor-165  and BMP-2 , as well as the basement membrane protein, laminin . Domain II has homology to the low-density lipoprotein receptor and was expressed as a complete and correctly folded domain in HEK-293 cells . The positions of the cysteine residues are strictly conserved suggesting that they are important for the tertiary structure of this domain. Domain III shares homology with structures present in the laminin α and γ chains as well as possessing EGF-like motifs and has been expressed in E. coli in its entirety  and as three separate, but overlapping, recombinant fragments in HEK-293 cells . The highest affinity binding of platelet-derived growth factor BB isoform has been mapped to the laminin-type IV and laminin type EGF-like repeat 3 regions of this domain . Despite the presence of an RGD motif in one of them, none of the recombinant fragments interacted with either α5β1 or αvβ3 integrins and no cell binding to any of the recombinant fragments could be demonstrated. This is in contrast to the same domain expressed in HT-1080 cells in which cells were shown to bind in an RGD-dependent fashion . A recombinant fragment from this domain was shown to bind to WARP, which is an ECM molecule expressed by chondrocytes that is a member of the von Willebrand factor A domain superfamily . Domain IV is the largest of the domains and consists of multiple IgG modules similar to those found in the neural cell-adhesion molecule. The number of the IgG motifs varies between species with the human perlecan sequence possessing 21 motifs, whereas the mouse perlecan sequence contains 14 . This domain has also been expressed as overlapping fragments, which have been shown to bind to the basement membrane nidogens, fibronectin and fibulin-2 [138, 139]. Domain V is the most C-terminal of the domains present in the protein core, and is made up of three regions with homology to the globular domains of the laminin α chain, which is interspersed with four epidermal growth factor-like repeats. The mouse sequence for domain V was expressed in HEK-293 cells and was decorated with either HS or CS [140-142]. The human sequence representing domain V (Leu3611 to Ser4391) was expressed in HEK-293 cells and also shown to be decorated with either HS or CS , whereas the recombinant product known as endorepellin (Glu3687 to Ser4391) when transfected into the same HEK-293 cells was expressed without GAG chains  suggesting that the missing 76 amino acids may be important for correct glycosylation at this site or that the glycosylation of recombinant PGs is variable and dependent on the expression system. Interestingly, when the sequence from domain V of the Drosophila melanogaster homologue of perlecan, unc-52, was expressed in HEK-293 cells, it was also produced as a protein without GAG decoration . The presence of GAG chains on these recombinant products may provide insights into the role of the perlecan molecule given that the interaction of recombinant domain V with nidogen, fibulin , laminin  or PRELP , an ECM protein that binds to collagen and is involved in matrix assembly, are all dependent on the presence of the GAG chains. By contrast, interactions with either α-dystroglycan  or the α2β1 integrin  have both been shown to be protein core dependent. Interestingly, removal of the GAG chains from recombinant domain V has been shown to enhance cell binding, suggesting that the presence of the carbohydrate inhibits the interaction with the integrin .