Perlecan (PLC) also known as basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2 (HSPG2), is a protein that in humans is encoded by the HSPG2 gene. The HSPG2 gene codes for a 4,391 amino acid protein with a molecular weight of 468,829. It is one of the largest known proteins. The name perlecan comes from its appearance as a "string of pearls" in rotary shadowed images.
Perlecan was originally isolated from a tumor cell line and shown to be present in all native basement membranes. Perlecan is a large multidomain (five domains, labeled I-V) proteoglycan that binds to and cross-links many extracellular matrix (ECM) components and cell-surface molecules. Perlecan is synthesized by both vascular endothelial and smooth muscle cells and deposited in the extracellular matrix of parahoxozoans.Aaron L Fidler, Carl E Darris, Sergei V Chetyrkin, Vadim K Pedchenko, Sergei P Boudko, Kyle L Brown, W Gray Jerome, Julie K Hudson, Antonis Rokas, Billy G Hudson (2017) Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues eLife 6:e24176 https://doi.org/10.7554/eLife.24176 Perlecan is highly conserved across species and the available data indicate that it has evolved from ancient ancestors by gene duplication and exon shuffling.
Structure
Perlecan consists of a core protein of molecular weight 469 kDa to which three long chains (each approximately 70-100 kDa) of glycosaminoglycans (often heparan sulfate, HS, but can be chondroitin sulfate, CS) are attached. The core protein consists of five distinct structural domains. The N-terminal domain I (aa ~1-195) contains attachment sites for HS chains. Although HS chains are not required for correct folding and secretion of the protein, lack of HS or decreased sulfation can decrease perlecan's ability to interact with matrix proteins. Removal of HS chains may affect matrix organization and endothelial barrier function. Domain II comprises four repeats homologous to the ligand-binding portion of the LDL receptor with six conserved cysteine residues and a pentapeptide, DGSDE, which mediates ligand binding by the LDL receptor. Domain III has homology to the domain IVa and IVb of laminin. Domain IV consists of a series of IG modules. The structure of only the 3rd immunoglobulin (IG)-like repeat in domain IV is shown in the figure labeled "HSPG2". The C-terminal Domain V, which has homology to the G domain of the long arm of laminin, is responsible for self-assembly and may be important for basement membrane formation in vivo. Domain V also has attachment sites for HS/CS chains. Thus, perlecan core protein and HS chains could modulate matrix assembly, cell proliferation, lipoprotein binding and cell adhesion.
Function
Perlecan is a key component of the extracellular matrix of cartilage where it is essential for normal growth plate development and long bone growth. The dwarfism exhibited by the perlecan null mouse resembles the phenotype produced by activating mutations in the gene for FGFR3, a receptor for fibroblast growth factors. Perlecan binds to growth factors involved in growth plate development. Perlecan isolated from developing growth plates has been shown to bind to FGF-2 via its heparan sulfate side chains, and to FGF-18 via domain III of its core protein and mediates their action on FGF receptors. Perlecan likely plays a critical role in the sequestration and/or delivery of FGF-2 and FGF-18 during endochondral ossification.
Perlecan is also a key component of the vascular extracellular matrix, where it interacts with a variety of other matrix components and helps to maintain the endothelial barrier function. Perlecan is a potent inhibitor of smooth muscle cell proliferation and is thus thought to help maintain vascular homeostasis. Perlecan can also promote growth factor (e.g., FGF-2) activity and thus stimulate endothelial growth and re-generation.
Modification of glycosaminoglycan chains
Modifications of the heparan sulfate chains on C- and N-terminal domains are the best-studied differences in the secretory pathway of perlecan. Chondroitin sulfate can be substituted for heparan sulfate, and sulfate incorporation or the sugar composition of the chains can change. Loss of enzymes involved in the heparan sulfate synthetic pathway lead to a number of conditions.
Differential heparan sulfate chain modification can occur through a number of regulatory signals. Perlecan in the growth plate of mouse long bones shows glycosylation changes in the chondrocyte progression from the resting zone to the proliferating zone. Although initially the glycosaminoglycan (GAG) chains of perlecan were thought to be exclusively heparan sulfate, chondroitin sulfate chains can be substituted and this may be dependent upon the cell type. By expressing a recombinant form of the N-terminal domain I of the protein and demonstrating that digestion of the peptide with either heparanase or chondroitinase did not lead to complete loss of the peptide's activity, it was shown that chondroitin sulfate chains can be added to human perlecan. This was in agreement with previous data showing chondroitin sulfate GAG chains attached to bovine perlecan produced by chondrocytes and that recombinant human domain I protein was glycosylated with both heparan and chondroitin sulfate chains when expressed in Chinese Hamster Ovary cells. The preferential addition of heparan sulfate or chondroitin sulfate chains to domains I and V could have an effect on the differentiation of mesenchymal tissues into cartilage, bone or any number of tissues, but the regulatory mechanism of changing from heparan sulfate to chondroitin sulfate addition are not well understood.
While studying the effect of proteoglycan composition on nephritic permselectivity, it was noted that puromycin treatment of human glomerular endothelial cells (HGEC) altered the sulfation level of GAG chains on proteoglycans such as perlecan, which in turn caused a decrease in the stability of the GAG chains. The core protein mRNA levels of proteoglycans were not affected, thus the decrease in GAG chains was as a result of some other factor, which in this case turned out to be a decrease in expression of sulfate transferase enzymes, which play a key role in GAG biosynthesis. It seems that there may be some overlap in diseases stemming from loss of heparan sulfate proteoglycan expression and loss of enzymes involved in heparan sulfate biosynthesis.
Degradation
Cells can modify their extracellular matrix and basement membranes in response to signals or stress. Specific proteases act on the protein in the extracellular environment when cells have a reason to move or change their surroundings. Cathepsin S is a cysteine protease that moderately attenuates binding of FGF-positive cells to a perlecan-positive substrate. Cathepsin S is a potential protease that acts on the core protein of perlecan in the basement membrane or stroma.
The heparan sulfate chains of perlecan bind growth factors in the ECM, and serve as co-ligands or ligand enhancers when bound to receptors. Another study showed that release of HS-bound basic FGF in culture could be achieved through treatment with stromelysin, heparitinase I, rat collagenase and plasmin, and these proteolysis sites are illustrated in figure 1. This was proposed as a non-exhaustive list of the proteases that could mediate release of growth factors from the heparan sulfate chains of perlecan. Although Whitelock et al. suggested that thrombin cleavage consensus sequences exist in the core protein of perlecan, they also postulate that any thrombin activation of perlecan actually comes from cleavage of other ECM constituents. This article states that heparanase is responsible for cleavage of the heparan sulfate chains of perlecan in matrix. This releases growth factors bound to the heparan sulfate, specifically FGF-10. Addition of heparanase to cell culture of epithelia in basement membrane caused an increase in epithelial cell proliferation due to FGF-10 release.
In a model of explant growth in vitro using corneal epithelium, Matrix Metalloproteinase (MMP) 2 expression correlates with an initial degradation of the original basement membrane. Reformation of basement membrane in culture was dependent on an initial upregulation followed by a downregulation of MMP-9, in contrast to the constant expression of MMP-2. This is not evidence that MMP-2 and MMP-9 directly cleave perlecan protein in vivo but shows that the proteins clearly modulate some factor in maturation of basement membrane. Another family of metalloproteases, the Bone Morphogenetic Protein 1/Tolloid-like family, releases the c-terminal endorepellin domain of the perlecan core protein. The laminin-like globular domain contains the active motif of endorepellin, and is unable to be cleaved by cells expressing mutant and inactive forms of the BMP-1 proteins. Furthermore, the critical residue necessary for this cleavage to take place was localized to Asp4197. This proteolytic process may have significance in disease as a corresponding fragment was found in the urine of patients suffering end-stage renal failure and in the amniotic fluid of pregnant women who have undergone premature rupture of the membrane.
Expression
Expression during development
Timing of gene expression during development varies from tissue to tissue. Basement membranes are often the driving force behind separating epithelia from stroma and connective tissue. Perlecan is of particular importance in cardiovascular, neural and cartilaginous development.
Pre-implantation blastocyst development is a controlled cascade of gene regulation and intercellular signaling. Extracellular perlecan has been observed at the blastocyst stage of mouse embryonic development, specifically upregulated at the point when the embryo reaches “attachment competence”. This finding was upheld at both the mRNA level and the protein level, shown by RT-PCR and immunostaining. Later embryonic development is just as precisely regulated as pre-implantation development, and is more complicated due to differentiation of all tissues. The first study of perlecan expression during embryonal development found that the protein was first expressed during development of the cardiovascular system, and later correlates with maturation of the majority of tissues in the body, i.e. separation of epithelial layers from endothelia and stroma by basement membranes. Again, this upregulation during cardiovascular development is concomitant with the role of perlecan's C-terminus as endorepellin.
Spatio-temporal specificity in trans-activation of the perlecan gene during development is key to the maturation of basement membranes and thus to the complete separation of epithelia from endothelia and stroma. A thorough study of perlecan expression during chick embryo development has shown that perlecan is present at the morula stage and for the rest of development, although expression can be transient and precisely timed in certain tissue predecessors. In the rat embryo, perlecan expression has been shown to increase in vascular smooth muscle cells (VSMCs) post e19 in fetal development. This correlates perfectly with the ceasing of proliferation of VSMCs at e18 and a change in their phenotype. The theory put forward in this study is that perlecan plays an anti-proliferative role for VSMCs once a certain developmental point is reached, much like confluence-dependent expression of perlecan in culture. These findings were corroborated by similar results from studies of rat pulmonary artery and lung epithelia. These tissues also were found to begin perlecan production once cell division had ceased, around fetal day 19.