**2. Structure and characteristics of CD146**

## **2.1. Genomic description**

The specific location of the CD146 gene is on the arm q23.3 of the chromosome 11 in humans and on the chromosome 9 in mice (www.ensembl.org). The gene encoding the CD146 protein extends over 14 kb. It consists of five immunoglobulin-like domains, two variable domains, and three constant domains of C2 type, as well as a transmembrane domain and an intracytoplasmic portion [6]. The extracellular part of the molecule, including the five immunoglobulin domains, is encoded by 13 exons; the transmembrane domain and the intracellular domain are encoded by three exons.

The promoter of CD146 presents different putative binding sites and motifs including AP1, AP-2, CRE, SP1, CArG, and c-myb. Analysis of this DNA segments suggests that the four SP-1 sites, the two AP-2 domains, and one response element to AMPc-(CRE) form the minimal promotor of CD146 [7]. Specific sites play a role in CD146 expression. The AP-2 sites, which are located at −131 and −302 by relative to the initial ATG, inhibit the expression of CD146 by 70 and 44%, respectively. Moreover, when mutated, the CRE site inhibits by 70% the transcription of the genes. Therefore, AP-2 [8] and CRE sites [9] have been described to modulate CD146 expression in melanoma cells, leading to an increase in tumor growth and metastatic potential in these cancers. In fact, the AP-2 binding site located in the promoter (located at −23 bp) is an inhibitor of the transcription of CD146 while the other AP-2 sites (located at −131 and −302, respectively) are transcription activators [8].

The size of CD146 mRNA is around 3.3 kb and has been first identified in human melanoma cancer cells [10]. Its encoding region is about 1940 bp. A large homology in the mRNA sequences exists between human and mouse, but differences can be noted. Thus, in humans, there is a lengthening of the 3' and 5' UTR region as wells as a loss of 6 pb in exon 2. The encoding regions and 5'UTR have a homology of about 80 and 72%, respectively, between the murine and human genes and there is only 31% of homology for the 3'UTR fragment. Finally, the protein sequence shares about 76% of homology between these two species [1, 10, 11].

## **2.2. Proteic structure and isoforms**

isoforms and that is present on different cell types could hence constitute a novel target for therapy. Different reviews have underlined its structural features, localization, and functions in the endothelium. This chapter thus mainly addresses the differences in CD146 isoforms with a special focus on their role in angiogenesis and the therapeutic tools targeting the molecule. Historically, CD146 was discovered in 1987 by Professor J.P. Jonhson for the first time. It was identified as a marker of melanoma progression. These data were obtained by using an antibody generated by mouse immunization with a cell lysate of metastasizing melanoma. This antibody (MUC18) allowed the identification of a 113 kDa transmembrane protein. MCAM (melanoma cell adhesion molecule) described as a marker of metastasizing melanoma [1].

In 1991, the team of Professor. F. Dignat-George identified Sendo-1 antigen as a marker of circulating endothelial cells in the blood by flow cytometry. This was made possible through the generation of a mouse monoclonal antibody named Sendo -1 [2] obtained by mice immunization with a HUVEC cell lysate. Sendo-1 was able to stain the human endothelium whatever the vessel size and its anatomical location within the vascular tree [3, 4]. Gicerin and

As reported in Kobé in 1997, CD146 (cluster of differentiation 146) is now the official name grouping Sendo-1/MUC18/MCAM/gicerin/HEMCAM (Sendo-MUC18 preCD, Workshop

The specific location of the CD146 gene is on the arm q23.3 of the chromosome 11 in humans and on the chromosome 9 in mice (www.ensembl.org). The gene encoding the CD146 protein extends over 14 kb. It consists of five immunoglobulin-like domains, two variable domains, and three constant domains of C2 type, as well as a transmembrane domain and an intracytoplasmic portion [6]. The extracellular part of the molecule, including the five immunoglobulin domains, is encoded by 13 exons; the transmembrane domain and the intracellular domain

The promoter of CD146 presents different putative binding sites and motifs including AP1, AP-2, CRE, SP1, CArG, and c-myb. Analysis of this DNA segments suggests that the four SP-1 sites, the two AP-2 domains, and one response element to AMPc-(CRE) form the minimal promotor of CD146 [7]. Specific sites play a role in CD146 expression. The AP-2 sites, which are located at −131 and −302 by relative to the initial ATG, inhibit the expression of CD146 by 70 and 44%, respectively. Moreover, when mutated, the CRE site inhibits by 70% the transcription of the genes. Therefore, AP-2 [8] and CRE sites [9] have been described to modulate CD146 expression in melanoma cells, leading to an increase in tumor growth and metastatic potential in these cancers. In fact, the AP-2 binding site located in the promoter (located at −23 bp) is an inhibitor of the transcription of CD146 while the other AP-2 sites (located at −131

HEMCAM refer both to the avian homologues of the molecule [5].

430 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**2. Structure and characteristics of CD146**

and −302, respectively) are transcription activators [8].

Report).

**2.1. Genomic description**

are encoded by three exons.

The proteic structure of CD146 is composed of a signal peptide of 28 amino acids (AA), five immunoglobulin domains (including two variable domains and three constant domains), a hydrophobic transmembrane region (AA 561–585), and an intracellular region. The protein sequence derived from the coding region of CD146 has a theoretical molecular weight of about 72 kDa. However, CD146 has a molecular weight of about 113 kDa. This difference is due to the glycosylation sites present on the protein sequence. Indeed, glycosylations represent about 35% of the total molecular weight of CD146 with mainly N-glycosylations. The presence of sialylation has also been shown [12].

CD146 has many similarities with other immunoglobulin family members such as BCAM (B-cell adhesion molecule) and ALCAM (activated leukocyte cell adhesion molecule), including the same number of immunoglobulin-like domains, similarity of functions and expression on tumor and endothelial cells. Thus, the ALCAM protein plays a role in CD4+ T lymphocytes and in tumor invasion [13, 14].

A short and a long isoform generated by alternative splicing have been identified as the two isoforms of membrane CD146. They have not been identified simultaneously. The long isoform was the first discovered in human melanocytes in 1987 and the short isoform was identified as a complementary DNA from chicken more recently [15]. In addition, a soluble form of CD146 was also identified in endothelial cell culture supernatant (HUVEC) and in bloodstream in patient [16].

Concerning the extracellular sequence, it is common to both isoforms. The difference is located in the intracytoplasmic portion [15]. The two isoforms are the result of an alternative splicing on exon 15 causing a reading frame shift. The short isoform displays a shorter intracytoplasmic domain containing a phosphorylation site for protein kinase C (PKC) and an interaction site for the protein with PDZ domain while the long isoform displays two domains for phosphorylation by PKC and an endocytosis signal sequence [15].

The intracytoplasmic domain sequence is similar to mice and human at 95 and 93% for the short isoform and long isoform, respectively. This conservation across species is in accordance with the important functions carried by the intracytoplasmic domain of CD146.

Finally, a soluble CD146 isoform with a molecular mass around 100 kDa, was identified for the first time in 1998 [16]. This isoform is detectable in human plasma and serum [17] and is generated by a metalloprotease-dependant shedding of the extracellular domain of CD146. The use of nonspecific inhibitors of metalloproteinase (GM6001) inhibits the formation of soluble CD146 [18].
