Structural Biochemistry/CCN Proteins

Introduction to the CCN Family
The CCN family consists of six secreted extracellular matrix associated proteins (CCN1 – CCN6). The CCN family was named as an acronym of the names of the first three CCN proteins discovered: Cyr61 (cysteine rich protein 61), CTGF (Connective Tissue Growth Factor), and NOV (nephoroblastoma overexpressed gene). The CCN family acts as key regulator of the ECM components and as signaling molecules involve in a variety of important biological functions. This includes adhesion and extracellular matrix remodeling, skeletal development and chrondrogenesis, angiogenesis and wound repair, and regulation of cell proliferation.

CCN Structure and Domains
In terms of structure, contains an N-terminal secretory signal peptide, four similar functional domains, the same organization, a common intron/extron pattern, a similar primary structure (a 40 – 60% similarity). All six CCNs have five exons, with the first exon corresponds to a signal sequence and the rest a discrete protein module. The CCN family members feature four functional domains: 1)An insulin-like growth factor bind protein-like module (IGFBP), 2) a von Willebrand factor type C repeat module (VWC), 3) a thrombospondin type-1 repeat module, and 4) a cysteine-knot-containing module (except CCN5). Each domain is separated by linker regions which are susceptible to proteolysis. Proteolysis acts as a regulator of CCN protein activity by cleaving the linker regions resulting in the production of truncated molecules and individual modules. The six members also contain 38 conserved cysteine residues which vary in length and composition throughout the family. These residues are located right after the VWC domain and acts as a hinge between the first and second half of the protein.

IGFBP: The IGFBP family consists of IGFBPs that have a high affinity to and bind to insulin-like growth factors. This allows them to control the transport, localization and metabolic breakdown of the IGFs. IGFBP domains are typically multidomain proteins with distinct cysteine-rich N- and C-terminal domains linked by variable linker region. The N-terminal domains are globular in structure and have 12 conserved cysteine residues while the C-termianl domain has 6 conserved cysteine residues. N- and C –domain work together in concert to contain and bind IGF molecules with high affinity.

CCN family’s IGFBP domain shares strong sequence similarity to the N-terminal domain of traditional IGFBP but lacks the C-terminal domain and thus binds IGF quite poorly. Scientists have created a model of the CCN’s IGFBP domain using a CPH model. To construct the model they used the 80 amino acid sequence residue of the N-terminal domain that both CCN and IGFBP shared 30% sequence identity with. The sequence is L-shaped and divided into two subdomains connected by a short stretch of coil. The first subdomain has a semi-folded with two-stranded antiparallel beta-sheets and two parallel loops. The loops are stabilized by a series of disulphide bonds which form a flat plane with the beta sheets. The second subdomain is a globular domain containing the IGF-binding site and is surrounded by a three-stranded anti-parallel beta-sheet stabilized by disulphide bonds.

VWC:The von Willebrand factor C repeat domain contains a 70-100 amino acid sequence motif which is constantly conserved in most ECM proteins. The VWC domain can be repeated multiple times in a single protein, increasing its activity, but vary in growth factor affinity. This difference among proteins is through to be a means of regulation, accounting for the varying substrate specificity not only in CCN proteins but in others as well. One of the functions of the domain is regulating bone morphogenic proteins, which facilitate bone, cartilage and organ growth, along regulating TFG-beta signaling.

The VWC domain in CCN proteins is made up of two sections. The upper section is made up beta-sheets while the lower section made up of fibronectin-like material held together by disulphide bonds. All the CCN proteins, except CCN6, have the VWC domain’s conserved cysteines. It results in a binding pattern of two disulphide bonds followed by the beta-sheets and then three more disulfide bonds. While the cysteines are conserved in the CCN proteins, their VWC domains vary in electrostatic surfaces. CCN1 and CCN5 have mostly negatively charged surfaces, CCN4 is mostly positively charged, and the rest are a mix of surface charges.

VWC repeats in most proteins functions to regular bone morphogenic proteins (BMP) and TGF-beta signaling. Both BMP and TGF-beta work closely with CCN proteins. BMPs influence bone and cartilage growth, both of which when damaged results in TGF-beta facilitating increased expressions of CCN 1, CCN 2 and CCN 5 with decreased CCN 4 expression.

TSP-1: This domain is made up of a 55 amino acid sequence residues. It has three distinct domain repeats: TSR-1 repeat, three epidermal growth factor-like repeats and seven aspartic acid-rich repeats, all inside a linear structure. The TSR-1 repeat is commonly found inside the human genome as well as in other eukaryotic organisms. It is composed mainly of small three-stranded anti-parallel beta-sheets organized into a right-helical shape and mainly functions as cell attachment sites in signaling and adhesion, inhibition of angiogenesis (blood cell growth), protein-binding sites for various growth factors and other ECM proteins, and glycosaminoglycan-binding sites inside the TSP-1 domain. Similarly to VWC many TSRs bind TGF-beta, however the TSP-1 domain in CCN proteins lack the necessary RFK tripeptide sequence to perform the binding. The TSP-1 domain does however share a 60 amino acid sequence similarity with the TSR superfamilies as well as the conserved cysteine residues, CSxTCG motif, arginine and tryptophan residues at the N-terminal of the domain.

Unlike the original, the TSP domain in the CCN proteins has fewer CWR layers, residues that form hydrogen bonds, along the domain’s three-stranded anti-parallel beta-sheet. Because of this they form fewer hydrogen bonds and only have a one tryptophan and two aginine residues. Three disulfide bonds are all present in the domain and hold the loops together around the beta-sheets. A positively charged domain surface is conserved in all six CCN proteins. Due to the ability of the TSP-1 domain to bind to glycoconjugates and its inhibition of angiogenesis, it may account for CCN proteins’ management of angiogenesis as well as their interactions with the ECM itself. CCN proteins, particularly CCN 1 and CCN 2, interaction with TGF-beta which facilitates their expressions, such in osteoblasts during bone fractures or breaks as well as to mediate angiogenesis. Their interaction with TGF-beta may be coordinated by their TSP-1 domain since original TSP domains also interact with TFG-beta. Mutant and missing TSP domains in CCN proteins are thought to be involved in the formation of colorectal and gastric carcinomas and Wilm’s tumours.

CT: The CT domain contains a cysteine knot motif made up of six conserved cysteine residues. It is thought to mediate many of the CCN protein functions which can be heterodimeric, where both the CT domains of CCN 2 and CCN 3 interact in glutathione S-transferase pull assays, and is involved in heparin binding, one of the components of ECM. All CCN protein CT domains contain a collection of positively charged residues which surround the beta-sheet loops with the heparin-binding site at the N-terminal portion of their domain.One of the main functions of the CCN proteins is regulation and remodeling of the ECM as well as adhesion. Because TSP domains are thought to bind heparin sulphated proteoglycans, scientist think that CT and TSP domains may work in tandem by directing how CCN proteins control and manipulate adhesion processes and ECM composition. For example, in order to induce adhesion in vascular smooth muscle cells, CCN 3 interacts with the integrin cell surface receptors and heparin sulphate proeoglycans.

The CT domain also contains a cysteine knot that is made up of a ring of eight residues linked by two disulphide bonds with a third bond going through the center of the knot. Next to the 3D knot are two two-stranded anti-parallel beta-strands. It is the domain’s cysteine knot motif that scientist think allows it to act as a dimerization molecule similar to those of growth factors. In NGF and TGF-beta, it is the disulphide bond in the center of the knot which helps direct dimerization. While all CCN proteins have similar arrangements, they all slightly differ in electrostatic surfaces. Scientists believed that this different in charge and their diverse sequences, not including their conserved cysteines, may account for the differences in CT domain ligands and binding partners.