Identification and Characterization of the Integrin α2β1 Binding Motif in Chondroadherin Mediating Cell Attachment

Abstract

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin ␣ 2 ␤ 1 and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same ␣ 2 ␤ 1 receptor. We identified a cell binding motif, CQLRGLRRWLEAK 318 by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK 318 remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the

Identification of potential biophysical and molecular signalling mechanisms underlying hyaluronic acid enhancement of cartilage formation

“…Because HA upregulated chondrogenic genes, such as collagen II and downregulated collagen I, it could be a potent chondrogenic agent for cartilage formation. Furthermore, the observed upregulation of chondroadherin, which increases extracellular signal-regulated kinase signalling [51], could create secondary signalling effects. Increased expression of syndecan-4, which binds to TGF-b1 via its heparan sulphate chains [52], also suggests that TGF-b1 and HA could potentially be applied simultaneously to produce synergistic effects.…”

Chondroadherin Fragmentation Mediated by the Protease HTRA1 Distinguishes Human Intervertebral Disc Degeneration from Normal Aging

“…It has the ability to bind triple helical collagen (5) and interact with cells via ␣ 2 ␤ 1 integrin (6) as well as via cell surface heparan sulfate proteoglycans (7). Interactions of CHAD with cells have been shown to lead to a variety of cellular responses with activation of intracellular signaling and changes in the cytoskeleton depending on which of the receptors alone or in combination are involved (7,8). CHAD clearly has the potential to influence cell metabolism and affect matrix homeostasis.…”

The C-Terminal Domain of Chondroadherin: A New Regulator of Osteoclast Motility Counteracting Bone Loss

“…(8) A synthetic cyclic peptide (cyclicCHAD) mimicked the structural constraints of this sequence in the intact protein and showed similar efficiency in inhibiting cell binding to CHAD. The unique peptide motif responsible for cellular binding was found to be primarily located in the octamer sequence LRRWLEAK(318) (8) (Supporting Fig. 2A).…”

“…Therefore, we hypothesized that this molecule could be involved in the regulation of bone metabolism. In previous work we had identified the C-terminal cell binding sequence of CHAD (8) that recognizes the a 2 b 1 integrin receptor. A synthetic cyclic peptide (cyclicCHAD) at cys-306 to cys-326 was selected for the present experiments because it exhibited similar efficiency in competitively inhibiting cell binding to CHAD and is likely to show better stability in biological systems.…”

“…The C-terminal binding sequence of CHAD is localized in one of the C-terminal disulfide loops. (8) An additional cell binding sequence is represented by the C-terminal peptide extension that binds heparan sulfate chains of cell surface proteoglycans with high affinity. (7) The complex system created by CHAD and collagen type II is assumed to be of functional importance for the communication between the cells and their surrounding matrix represents a component of bone matrix, where it promotes attachment of osteoblasts and binds to collagen with a higher affinity than osteonectin and decorin.…”