A novel peptide sequence in perlecan domain IV supports cell adhesion, spreading and FAK activation

Abstract

Perlecan/HSPG2 is a large, multi-domain, multifunctional heparan sulfate proteoglycan with a wide tissue distribution. With the exception of its unique domain I, each of perlecan's other four domains shares sequence similarity to other protein families including low density lipoprotein (LDL) receptor, laminin alpha chain, neural cell adhesion molecule (NCAM), immunoglobulin (Ig) superfamily members, and epidermal growth factor (EGF). Previous studies demonstrated that glycosaminoglycan-bearing perlecan domain I supports early chondrogenesis and growth factor delivery. Other sites in the core protein interact with other matrix molecules and support cell adhesion, although the peptide sequences involved remain unidentified. To identify novel functional motifs within perlecan, we used a bioinformatics approach to predict regions likely to be on the exterior of the folded protein. Unique hydrophilic sequences of about 18 amino acids were selected for testing in cell adhesion assays. A novel peptide sequence (TWSKVGGHLRPGIVQSG) from an immunoglobulin (Ig) repeat in domain IV supported rapid cell adhesion, spreading and focal adhesion kinase (FAK) activation when compared to other peptides, a randomly scrambled sequence of the domain IV peptide or a negative control protein. MG-63 human osteosarcoma cells, epithelial cells and multipotent C(3)H10T1/2 cells, but not bone marrow cells, rapidly, i.e., within 30 min, formed focal adhesions and assembled an actin cytoskeleton on domain IV peptide. Cell lines differentially adhered to the domain IV peptide, suggesting adhesion is receptor specific. Adhesion was divalent cation independent and heparin sensitive, a finding that may explain some previously poorly understood observations obtained with intact perlecan. Collectively, these studies demonstrate the feasibility of using bioinformatics-based strategies to identify novel functional motifs in matrix proteins such as perlecan.

Perlecan Domain IV Peptide Stimulates Salivary Gland Cell AssemblyIn Vitro

“…The intact perlecan molecule that is produced by cultured acinar cells is expected to be modified by heparan sulfate chains that in previous studies were found to conceal the adhesive domain IV motifs. 11 Fibroblasts cultured on intact perlecan failed to make stable focal adhesion contacts because of the antiadhesive activity of gagosylated perlecan domain I. 23 Consistent with this, our studies showed less FAK activation and, more strikingly, a failure to cluster phosphorylated FAK to sites of focal adhesions when cells were cultured on plastic.…”

“…The functionality of the cultured salivary acinar cells in vitro is confirmed by their secretion and organization of their own basement membrane including the critical component perlecan that can support further growth and differentiation by virtue of its ability to interact with cell adhesion molecules that can influence integrin-mediated events. 11,15 Uniquely, no non-human-derived material was needed for isolation or culture of the acinar cells.…”

“…Our earlier studies suggest that adhesion to PlnDIV peptide is mediated through nonintegrin receptors as inhibition of b1 integrins only partially affected adhesion to surfaces modified with the peptide. 11 The peptide's origin from an Ig loop on PlnDIV suggests that signaling could be induced via interactions with Ig superfamily members on the surfaces of the acinar cells through homophilic or heterophilic binding events. These interactions can initiate signaling events that alter cytoskeletal dynamics and organization leading to cell migration, differentiation, and ultimately acinar assembly.…”

“…Domain IV of perlecan (PlnDIV) contains a novel peptide sequence, which supports adhesion, spreading, and focal adhesion kinase (FAK) activation. 11 Additionally, PlnDIV contains immunoglobulin (Ig) repeats that are similar to those found in Ig superfamily members such as the neural cell adhesion molecule or the platelet endothelial cell adhesion molecule. 11 We tested the hypothesis that salivary gland cells cultured on PlnDIV peptide will receive the appropriate cues that allow them to differentiate and mimic their glandular phenotype.…”

“…11 Additionally, PlnDIV contains immunoglobulin (Ig) repeats that are similar to those found in Ig superfamily members such as the neural cell adhesion molecule or the platelet endothelial cell adhesion molecule. 11 We tested the hypothesis that salivary gland cells cultured on PlnDIV peptide will receive the appropriate cues that allow them to differentiate and mimic their glandular phenotype. We used PlnDIV peptide to promote attachment and subsequent differentiation of cultured human acinar cells into salivary units, a useful first step toward the culture of acinar cells free of animal products that can be implanted into patients.…”