[CANCER RESEARCH 55, 2140-2149, May 15, 1995] Identification of a New Immunoglobulin Superfamily Protein Expressed in Blood Vessels with a Heparin-binding Consensus Sequence Marie E. Beckner,1 Henry C. Krutzsch, Mary L. Stracke, Sonya T. Williams, Jorge A. Gallardo, and Lance A. Liotta Laboratory of Pathology, National Cancer Institute. N1H, Bethesda, Maryland 20892 ABSTRACT We have identified and sequenced cDNA from a human melanoma cell library that encodes a new protein, called AAMP, that shares A novel immunoglobulin-type protein expressed in blood vessels has characteristics with immunoglobulin superfamily proteins and con been identified. The cDNA for AAMP (angio-associated, migratory cell tains a heparin-binding consensus sequence. Immunoblotting and protein) was first isolated from a human melanoma cell line during a search for motility-associated cell surface proteins. Upon analysis of the immunohistochemistry using affinity-purified antibodies with speci tissue distribution of AAMP, it was found to be expressed strongly in ficity for rAAMP and for a derived peptide, P189, were used to endothelial cells, cytotrophoblasts, and poorly differentiated colon adeno- identify the native protein in human cultured cells and tissues. In carcinoma cells found in lymphatics. The sequence of AAMP predicts a addition, the heparin-binding capacity of peptide 189 was determined protein (M,. 49,000) with distant identity (25%) to known proteins. It in order to evaluate the potential role of this epitope in cell adhesion. contains immunoglobulin-like domains [one with multiple homologies to deleted in colon carcinoma (DCC) protein], the WD40 repeat motif, and a heparin-binding consensus sequence. A 1.6-kilobase niKNA transcript of MATERIALS AND METHODS AAMP is detected in tissue culture cell lines and tissues. Affinity-purified polyclonal antibodies, anti-recombinant AAMP, and anti-peptide 189 cDNA Library Screening. A human melanoma A2058 cDNA expression (AAMP derived) recognize a ,Ur 52,000 protein in human tissue and library (Agtll) was screened with an antibody, 1AA3AA, suspected to inhibit cellular extracts. The protein size is in keeping with the inKNA and cell motility. predicted sequence. The AAMP-derived peptide, P189, contains a hepa 1AA3AA Monoclonal Antibody Preparation. Mouse hybridoma clones rin-binding domain (dissociation constant, 14 pmol) and mediates hepa- were generated with A2058 melanoma cells using the myeloma cell line rin-sensitive cell adhesion. The shared expression of AAMP in endothelial X63.Ag8.653 (a generous gift of R. P. Siraganian, National Institute of Dental cells, trophoblasts, and tumor cells implies a common function in migrat Research, NIH, Bethesda, MD) (20-22). They were screened for inhibition of ing cells. melanoma chemotaxis (23) to autotaxin (24), type IV collagen (Collaborative Biomedicai Products, Bedford, MA), and laminin (a gift of S. Aznavoorian, National Cancer Institute, NIH, Bethesda, MD). INTRODUCTION DNA Sequencing. The Agtl 1 phage insert, AAMP, from the A2058 cDNA Molecules that mediate cell-cell and cell-substrate interactions in library, was subcloned into Bluescript plasmid (Stratagene, La Jolla, CA) for double-stranded DNA sequencing (both strands; Ref. 25). PCR products gen clude members of the immunoglobulin superfamily. These contain erated from the amino terminal region of AAMP cDNA (obtained with human immunoglobulin-like domains that share evolutionary homology and brain mRNA; Clontech, Palo Alto, CA) were sequenced with Taq polymerase function primarily in recognition and binding processes on cell sur (Perkin-Elmer Roche Molecular Systems, Branchburg, NJ) both directly and as faces (1-5). Most are cell surface proteins, a few are intracellular, and subcloned cDNA (26-28). some are secreted (4). Immunoglobulin superfamily proteins and the ALIGN Sequence Comparisons. Immunoglobulin domains (20 residues mechanisms involved in their regulation of migratory cells are of beyond each predicted cysteine bond) of immunoglobulin superfamily mem bers were ALIGN matched with AAMP (29) using standard parameters. Scores special interest. These proteins include those that help mediate endo of 3.1, 4.3, and 5.2 SD indicate probabilities of 10~3, 10~5, and 10~7, thelial cell interactions with lymphocytes, monocytes, neutrophils, and tumor cells that result in adhesion and transendothelial migration respectively, that unrelated domains show similarity by chance (30, 31). (6-9). Activation of T cells (10,11) and the processes of cell adhesion Recombinant AAMP Purification. AAMP insert was subcloned into pGEX-lÀT £coRI/BAP plasmid (Pharmacia, Uppsala, Sweden) for production and migration in the nervous system also involve immunoglobulin of AAMP protein fused with glutathione 5-transferase in Escherichia coli superfamily members (3). The binding mechanisms of some immu according to the manufacturer's instructions. Bacteria were induced with noglobulin superfamily proteins that participate in the above pro isopropyl-ß-D-thiogalactopyranoside, sonicated, and centrifuged. Supernates cesses, including PECAM,2 NCAM, CD4, as well as other adhesive were loaded onto glutathione Sepharose columns (Pharmacia), washed, and proteins such as CD44, involve glycosaminoglycans (heparin, chon- thrombin (Sigma Chemical Co., St. Louis, MO) digested for purification of droitan sulfate, and hyaluronan, in these examples) (10,12-18). Some recombinant AAMP from glutathione S-transferase. Digestion ( 1 h) occurred at immunoglobulin superfamily members are multifunctional and par a thrombin-sensitive site in AAMP, aa 14. ticipate in both cell binding and signaling (4, 19). Determining the Peptide Preparation. AAMP-derived peptides and variants were synthe identity and characteristics of new proteins that participate in cellular sized on a Biosearch model 9600 synthesizer (Bedford, MA) using standard Merrifield solid phase synthesis protocols and f-butoxycarbonyl chemistry and interactions involving migrating cells and endothelium should help were analyzed by reverse-phase HPLC. To generate peptides from recombi elucidate mechanisms involved in biological processes such as in nant AAMP, it was reduced and alkylated (AMsopropyliodoacetamide), di flammation, wound healing, and métastasesof tumor cells. gested with trypsin, and subjected to HPLC chromatography (32). Peptide Sequencing. Amino acid sequence analysis was performed using Received 9/26/94; accepted 3/2/95. a Porton/Beckman 2090 on-line sequencer (Fullerton, CA) using standard The costs of publication of this article were defrayed in part by the payment of page program no. 1. Phenylthiohydantoin amino acid analysis was carried out on a charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Beckman System Gold system (Fullerton, CA) using a modified sodium 1To whom requests for reprints should be addressed, at Laboratory of Pathology, Bldg. acetate gradient program and a Hewlett-Packard narrow bore C18 column. 10, Rm. 2A-33, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD Polyclonal Antibody Preparations. Polyclonal antipeptide antibodies spe 20892. 2 The abbreviations used are: PECAM, platelet-endothelial cell adhesion molecule; cific for AAMP (generated in rabbits using peptides conjugated to bovine NCAM, neural cell adhesion molecule; AAMP, angio-associated migratory cell protein; albumin) were affinity purified on columns of peptide covalently attached to rAAMP, recombinant AAMP; aa, amino acid(s); DPBS, Dulbecco's phosphate-buffered Affi-Gel 10 beads (Bio-Rad, Richmond CA). Polyclonal anti-recombinant saline; DMEM, Dulbecco's minimum essential medium; bp, base pair(s); kb, kilobase(s). AAMP antibodies were generated in rabbits against recombinant AAMP. 2140 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1995 American Association for Cancer Research. AAMP, A NEW VASCULAR PROTEIN Anti-rAAMP was affinity purified on columns of Affi-Gel 10 beads with human brain mRNA with gene-specific, nested primers using 5'- recombinant AAMP covalently attached. RACE PCR contain sequence that codes for known sequence of Human Tissue and Cell Lysate Preparations for Immunoblotting. AAMP and several additional 5' protein residues consistent with an Human tissues were homogenized in 3% SDS and 4% ß-mercaptoethanol open reading frame. Four subcloned PCR products code for aa 2-7, buffer at 100°C.Whole cell lysates of A2058 melanoma cells (passaged fewer followed by a "G" in the 5' direction. One of these products contains than 20 times) and bovine endothelial cells (passage 6), aortic and corneal a complete AUG codon at this site, consistent with an initiating (gifts of N. H. Guo and J. Kaiser, both from the National Cancer Institute, NIH, methionine. Several other products that also code for aa 2-7 show Bethesda, MD), were prepared in 0.5% NP40 buffer. Immunoblot Preparation. Tissue and cell lysates electrophoresed in HV/c alternative sequence (<40 bp) replacing the AUG codon at this loca SDS polyacrylamide gels were blotted and reacted with either affinity-purified tion that is currently being characterized. The reactivity of the anti- polyclonal rabbit anti-rAAMP or anti-P189 (1-2 fig/ml) and then goat anti- peptide antibody generated to P189 (aa 14-25) with the natural rabbit IgG. Blots were developed with diaminobenzidine. Competition studies protein (Fig. 2) indicates that coding message for protein is present in used peptide and recombinant