Elsevier

Gene

Volume 327, Issue 2, 3 March 2004, Pages 171-183
Gene

CD109 represents a novel branch of the α2-macroglobulin/complement gene family

https://doi.org/10.1016/j.gene.2003.11.025Get rights and content

Abstract

We report here the genomic organization and phylogenic relationships of CD109, a member of the the α2-macroglobulin/complement (AMCOM) gene family. CD109 is a GPI-linked glycoprotein expressed on endothelial cells, platelets, activated T-cells, and a wide variety of tumors. We cloned full-length CD109 cDNA from the mammalian U373 cell line by RT-PCR and performed analysis of its corresponding genomic sequence. The CD109 cDNA spans 128 kb of chromosome 6q with its 33 exons constituting approximately 3.3% of the total CD109 genomic sequence. Sequence analysis revealed that CD109 contains specific motifs in its N-terminus, that are highly conserved in all AMCOM members. CD109 also shares motifs with certain other AMCOM members including: (1) a thioester ‘GCGEQ” motif, (2) a furin site of four positively charged amino acids, and (3) a double tyrosine near the C-terminus. Based on a phylogenic analysis of human CD109 with other human homologs as well as orthologs from other mammalian species, C. elegans (ZK337.1) and E. coli homologs, we propose CD109 represents a novel and independent branch of the α2-macroglobulin/complement gene family (AMCOM) and may be its oldest member.

Introduction

Innate immunity is a multifactorial response against invading pathogens that employs defense mechanisms that do not require prior immunologic exposure to be effective. One family of proteins with a significant role in innate immunity includes the α2 macroglobulin/complement gene family of proteins (AMCOMs) (Sunyer and Lambris, 1998).

In humans, seven members of the AMCOM family have now been cloned. These include α2-macroglobulin, pregnancy zone protein (Kan et al., 1985), complement proteins C3 (Fong et al., 1990), C4 (Moon et al., 1981), C5 (Lundwall et al., 1985), KIAA1283 (Nagase et al., 1999), and CD109 (as shown herein and in a prior study; Lin et al., 2002). Despite having distinct roles and functions in the innate immune system, four of these proteins, α2-macroglobulin, C3, C4 and C5, share a common mechanism of action that includes covalent binding to substrate through thioester transacylation and clearance of immune complexes Tack, 1983, Dodds and Law, 1998. Although thioester sites are also present in both KIAA1283 and CD109, their mechanisms of action and biological roles have not yet been elucidated.

α2-macroglobulin is synthesized as a soluble single chain, constitutive protease inhibitor, as is the closely related pregnancy zone protein (PZP), a protein upregulated during pregnancy. In contrast, the complement C3, C4 and C5 proteins, which are also soluble, are synthesized as single chain molecules that undergo a series of post-translational modifications to transform into two polypeptides held together with disulphide bridges Chan and Atkinson, 1983, Anthony et al., 1985, Lundwall et al., 1985. CD109, on the other hand, is a membrane bound GPI anchored molecule, which appears to consist of three protein antigens of 120, 150, 180 kDa Suciu-Foca et al., 1985, Brashem-Stein et al., 1988, Sutherland et al., 1991, Haregewoin et al., 1994, Lin et al., 2002. Although an explanation for the appearance of the 150 kDa form of the protein via autocatalytic cleavage of the 180 kDa form has been proposed (Lin et al., 2002) (in a biomechanism similar to that of AMCOM members), it is not at all clear if this mechanism accounts for all three forms of CD109 observed in earlier studies Suciu-Foca et al., 1985, Brashem-Stein et al., 1988, Sutherland et al., 1991, Haregewoin et al., 1994, nor may it be the only explanation for the 150 kDa form.

Previously we described CD109 as a (GPI)-anchored protein Haregewoin et al., 1994, Solomon et al., 1998 expressed on activated T-cells and platelets, and constitutively expressed on endothelial cells, as well as on many tumor cell lines. In that report we demonstrated that this antigen had been previously identified by Suciu-Foca et al. (1985) as the T-cell late differentiation antigen (LDA-1) and as a marker for activated T-cells and platelets Brashem-Stein et al., 1988, Sutherland et al., 1991. In our earlier report (Haregewoin et al., 1994) we also suggested that CD109 was the same as the Gov antigen, a marker which had been previously described as an allo antigen on platelets (Kelton et al., 1990). This was later confirmed by Kelton et al. (1998) and work done at the 5th Leukocyte Workshop determined that antibodies to this antigen defined a new cluster of differentiation, i.e. CD, and in 1995 the antigen was named CDw109 (and later in 1998 renamed CD109) Sutherland and Yeo, 1995, Sutherland and Yeo, 1998.

Here we report the N-terminal amino acid sequences of the CD109 180 and 150 kDa forms. Using this information in combination with bioinformatics we cloned full length CD109 cDNA from the U373 astroglioblastoma cell line. We then undertook the analysis of the organization of the CD109 gene. Based on genomic organization, as well as cDNA and translated protein sequences we are able to reveal that CD109 is a phylogeneticly independent, yet related member of the α2 macroglobulin/complement C3, C4, C5 gene family. The sequence obtained is essentially identical to the earlier cloned CD109 from KG1a cells that contains the Gov b epitope (Schuh et al., 2002).

Through sequence comparison of CD109 with EST (expressed sequence tag) orthologs from mouse, cow and rat as well as a homolog from nematode we map key blocks of conserved amino acids as we propose that CD109 is a novel, distinct, and apparently the oldest known member of the ancient AMCOM gene family.

Section snippets

Cell lines

The U373 and HPB-ALL cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), Chinese Hamster Ovary (CHO Kia) cell line from D. Golenbock (Worcester) and MDA 231 from P. Hauschka (Boston). The U373 and CHO cell lines were cultured in Cellgro Hams-F12 media (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Gemini, Woodland, CA), 2 mM l-glutamine (Sigma, St. Louis, MO), and penicillin/streptomycin (Sigma) at 37 °C and 5% CO2. The HPB-ALL cells

Sequencing of the CD109 N-terminus

To determine the amino acid sequences of individual CD109 species (i.e. 180, 150 kDa bands), we immunopurified both species of the protein from human cell lines and platelets (Solomon, 1996). After purification (see Materials and methods) Edman degradation based sequencing of the N-terminus was carried out for both 180 and 150 kDa species (Table 1). Homology based searches using the NCBI databases showed that the peptide sequences we had obtained for the 180 and 150 kDa species of CD109 shared

Discussion

CD109 appears to be the most recently characterized member of the AMCOM family that also includes C3 (Fong et al., 1990), C4 (Moon et al., 1981), C5 (Lundwall et al., 1985), α2-macroglobulin (Kan et al., 1985), and KIAA1283 (Nagase et al., 1999). While CD109 has been the most recent to be discovered, in evolutionary terms it seems to have arisen from a “α2M-ancestral” protein through gene duplication similar to the gene duplication events that gave rise to the human C3 and KIAA1283 genes. In

Acknowledgements

This work was supported by NIH ROI grant number GM63244 (Robert Finberg, Parul Sharma and Melvin Chan) and Susan G. Komen Breast Cancer foundation (Keith Solomon). The authors thank Damon Milhem for technical advice during the course of this work. We also thank H. Saito (Dana Farber Cancer Institute, Boston, MA), for generous gift of several human placental and human activated T-cell phage libraries.

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    These authors contributed equally to this work.

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