The Protein Journal, Vol. 25, No. 4, June 2006 ( 2006) DOI: 10.

1007/s10930-006-9015-6 Published Online: September 1, 2006

Expression and Purication of Human PAG, a Transmembrane Adapter Protein Using an Insect Cell Expression System and its Structure Basis
Satoru Takeuchi1,2

In this study, we report the purication and structure basis of human phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a C-SRC tyrosine kinase (CSK)-binding protein. Human PAG was produced using an insect cell expression system. The PAG was puried by metal anity, ion exchange, and gel ltration chromatographies. The nal purity of gel-puried PAG was evaluated by SDS-PAGE and mass spectrometry. Recombinant human PAG migrates to 60 kDa on SDS-PAGE gel, while native PAG is a 46 kDa transmembrane adapter protein in lipid rafts. Recombinant human PAG has a dierence of 2590.7 Da with a calculated mass (47803.41 Da) and an observed mass (50394.1 Da) by mass spectrometry. Consequently, although human PAG sequence shares well-known sites for modications such as myristoylation, palmitoylation, and tyrosine phosphorylation sites, perhaps the dierence suggests the existence of unknown modication sites. We show the high PAG-binding ability with CSK in vitro as well as the human PAG structure characterized by 11 a-helix structures including a 3 kDa transmembrane domain.
KEY WORDS: SFKs; myocardial infarction; protein modication; mass spectrometry; a-helix structure.

1. INTRODUCTION SRC family tyrosine kinases (SFKs) play important roles in ligand-induced cellular responses including proliferation, survival, adhesion, and migration. The kinase activation of SRC in myocardial infarction is associated with the expansion of infarct volume and the decline of cardiac function including brosis and survival. Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) was found in lipid rafts as a SRC negative regulator or a C-SRC tyrosine kinase (CSK) activator (Brdicka et al., 2000; Kawabuchi et al., 2000; Takeuchi et al., 2000). This regulator may be appli-

cable as a novel approach for myocardial infarction treatment. In the present study, we describe its purication method when recombinant human PAG was puried via metal anity, ion exchange and gel ltration chromatographies. The nal purity of PAG was over 95% on SDS-PAGE and further evaluated by mass spectrometry. In addition to the purication method, the present study shows a whole structure of the PAG with a modied 50394.1 Da mass variously, which human PAG is composed of eleven a-helix structures including a 3 kDa transmembrane domain. And also, we show the high binding ability between PAG and CSK shown in the presence of 1.5 M NaCl.

Department of Protein Research, ProstaColon, 85 NE, Takamatsu, Kahoku, Ishikawa, 9291215, Japan. To whom correspondence should be addressed. E-mail: prostacolon@yahoo.co.jp

Abbreviations: SFKs, SRC family tyrosine kinases; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; CSK, C-SRC tyrosine kinase; GRP78, 78 kDa glucoseregulated protein; ORP150, 150 kDa oxygen-regulated protein.

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296 2. MATERIALS AND METHODS 2.1. Recombinant Baculovirus Construction, Expression and Purication of PAG The cDNA encoding human PAG (RZPD, EX-T0343-I01, N-His) was cloned into donor vector pFastBac HTa and recombinant pFasBac HTaPAG was transformed into competent DH10Bac E. coli cells. Recombinant Bacmid-PAG was transfected into Sf9 insect cells using the CELLFECTIN reagent based on the instruction manual for the Bac-to-Bac baculovirus expression system (Invitrogen). Recombinant human PAG was produced according to the above manual as follows. Sf9 insect cell lines were maintained in spinner asks (100 ml culture) at 27C from 30 104/ml to 250 104/ml for 34 days in IPL41 medium (Invitrogen) including 10% FBS. Sf9 insect cells were infected with recombinant baculovirus during a logphase of the cells, and then recombinant human PAG was expressed. Recombinant His-tagged PAG was puried in 50 mM Tris buer (pH7.4) including 0.5 M NaCl, imidazole ($0.4 M), 10% glycerol, 0.5% NP40, and 1 mM 2-ME using the TALON metal resin (Clontech) column. The PAG was eluted when imidazole was reached to a 0.2 M concentration. The collected active fractions were further puried in a nal buer (50 mM Tris pH7.4, 0.15 M NaCl, 0.5% NP40, and 1 mM 2-ME) via ion exchange (Bio-Rad, High Q Econo-Pac) and gel ltration (Amersham, Sephacryl S-400 HR) chromatographies. 2.2. MALDI-TOF (MS) of PAG Mass spectra of gel-puried PAG were obtained using a Voyager-DE STR BiospectrometryTM Workstation (PerSeptive Biosystems Inc., MA, USA); 1 ml of PAG solution was mixed with a fresh sinapinic acid matrix in 40% acetonitrile including 0.1% triuoroacetic acid on a MALDI plate. 2.3. Secondary Structure Prediction, Hydropathy Proling, and Sequence Alignment of PAG(s) A putative secondary structure of human PAG was obtained using the SOPM secondary structure prediction tool (Geourjon and Deleage, 1994). A hydropahy prole of human PAG sequence was

Takeuchi obtained using the Kyte-Doolittle tool (Kyte and Doolittle, 1982) on ExPASy server (http:// www.br.expasy.org/). The sequences of PAGs from the mouse, the rat, and the human were aligned using the DDBJ CLUSTALW 1.83 program (Thompson et al., 1994, Miyazaki et al., 2004). 2.4. CSK-binding Assay We previously described a purication method of recombinant human CSK (in submission). In this assay, we mixed Sf9 insect cells expressed the CSK with Sf9 insect cells expressed PAG. After metal anity and ion exchange chromatographies, active fractions were further gel-chromatographed using the same column under the dierent NaCl concentrations ranging from 0.15 to 1.5 M (buers: 50 mM Tris pH7.4, 0.15$1.5 M NaCl, 0.5% NP40, and 1 mM 2-ME).

3. RESULTS AND DISCUSSION 3.1. SDS-Page Analysis of PAG Human PAG was overexpressed in Sf9 insect cells after recombinant baculovirus infection (Fig. 1A). The gel-puried PAG has a high purity of more than 95% as shown in Fig. 1B. Native PAG is an approximately 46 kDa transmembrane adapter protein located in lipid rafts, which however migrates anomalously as an $80 kDa protein on SDSPAGE gels. In fact, recombinant His-tagged PAG was approx. 60 kDa on our analyzed SDS-PAGE gel (Fig. 1). Abundant acidic residues within protein sequence aect migration of the protein on SDSPAGE gel. Moreover, protein modication (e.g., myristoylation, glucosylation, palmitoylation, and phosphorylation etc.) is also similar. We think that recombinant His-tagged PAG may migrate from 46 to 60 kDa on our analyzed SDS-PAGE gel (Fig. 1B) because human PAG sequence indeed has the highly conserved myristoylation, palmitoylation, and tyrosine phosphorylation sites shown in Fig. 3B. 3.2. Mass Spectrometry and Modication of PAG Its actual observed mass was 50394.1 Da, no matching with the calculated mass, while a calculated mass of recombinant His-tagged PAG was 47803.41 Da (Fig. 2). A dierence with the

Expression and Purication of Human PAG

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Fig. 1. SDS-PAGE analysis of the expressed and gel-puried PAGs. (A) Lane 1: standard molecular weight marker; lane 2: PAG expression in Sf9 insect cells (crude extract). (B) Analysis of gel purication step of PAG. lane 1: standard molecular weight marker; other lanes: gel-puried PAGs.

calculated and observed masses of the PAG is 2590.7 Da. Even if we add the known modied masses of myristoylation (210 Da 1), palmitoylation (238 Da 2), and phosphorylation (80 Da 5) to a calculated mass of recombinant His-tagged PAG, the remaining dierence (1504.7 Da) is unknown. The phosphorylation of proteins, catalyzed by dierent kinases, is critical to cellular functions associated with signal transduction. This process involves a change in the phosphorylation status of downstream proteins by modication of specic tyrosine or serine/threonine residues. Human PAG shares ten tyrosine residues within its cytoplasmic domain sequence, nine of which are potential phosphorylation sites by SFKs (YXXV/I/L). PAG also

contains many threonine and serine residues, which are potential sites for phosphorylation (Fig. 3B). From a result of our mass spectrometry, we think that perhaps the PAG shares the unknown modication sites including potential modication sites for insect endogeneous kinases because an observed mass of the human PAG expressed in Sf9 insect cells is larger than its predicted mass. 3.3. PAG Structure The 3D structure of PAG is not yet claried. PAG is a 429 amino acid (aa) in the mouse, 424aa in the rat, and 432aa in the human; each including a short extracellular domain ($13aa), a transmembrane domain (27aa), and a large cytoplasmic domain. The human PAG shows over 90% similarity with the mouse and the rat in the amino acid sequence. These PAGs are also characterized by a common C-terminal TRL sequence which interacts with an N-terminal PDZ domain of ERM-binding phosphoprotein EBP50 (Fig. 3B) (Brdickova et al., 2001; Itoh et al., 2002). These ndings suggest that mammalian PAGs have a highly conserved
c Fig. 3. Molecular structure of PAG. (A) Putative secondary structure (top) and hydropathy prole (bottom) of human PAG were obtained using the SOPM secondary structure prediction and Kyte-Doolittle tools. (B) Sequence alignment of PAGs from the mouse, the rat, and the human were performed using the program DDBJ CLUSTALW 1.83. The following sequences were used for the alignment; a mouse (GenBank Accession No. Q3U1F9), a rat (Q9JM80), and a human (AF240634). Identical residues were hatched. Putative transmembrane domains were boxed. Representative known modication sites are as follows. The N-terminal myristoylation (glycine), palmitoylation (cysteine), and phosphorylation (tyrosine) sites were shown with asterisks, bolds, and arrow heads, respectively.

Fig. 2. Mass spectrometric analysis of gel-puried PAG. Purity of gel-puried PAG was nally evaluated by mass spectrometry. The masses observed are shown.

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Takeuchi

Expression and Purication of Human PAG

299 study showed a relation between SFK and ER chaperone, in which GRP78 (78 kDa glucose-regulated protein), an ER chaperone, regulates c-SRC kinase activity in vitro (Carlino et al., 1994). We have recently cloned two 150 kDa oxygen-regulated protein (ORP150) cDNAs (GenBank Accession No. DQ350134 and DQ372932) including transcript variant from cardiac tissues with infarction. ORP150 is specically induced by hypoxia stress (Kuwabara et al., 1996) and shows chaperone function in the ER. Presently, we are studying relations between ORP150 and activity regulation of SFKs with PAG. Inappropriate or deregulated phosphorylation is deeply associated with various diseases such as diabetes, cancer, neurological and autoimmune diseases, and heart disease including cardiac insuciency. A clear understanding of phosphorylation associated with a specic signaling pathway is the key to understanding pathway activation, related diseases and an ultimate approach.

Fig. 4. PAG-CSK complex formation. PAG and CSK were able to maintain their complex formation in the presence of 1.5 M NaCl. lane 1: standard molecular weight marker; other lanes: gelpuried PAG-CSK complexes.

3D structure. Unfortunately, we could not nd a similar structure to human PAG from the protein structure similarity search. However, we predicted a secondary structure of human PAG using the above tool with an entire accuracy of 70% (helix: 66.3%, sheet: 60.9%, and coil: 74%) (Geourjon and Deleage, 1994). We show the human PAG structure which is composed of eleven a-helix structures including a 3 kDa transmembrane domain claried by hydropahy proling (Fig. 3A). 3.4. PAG-CSK Complex Formation Under High Ionic Strength Condition Previous studies have shown a strong interaction between PAG and CSK (Kawabuchi et al., 2000). We investigated a complex formation of PAG-CSK under the various concentrations of NaCl ranging from 0.15 to 1.5 M NaCl. Surprisingly, this study showed that PAG and CSK were able to maintain their complex formation under a high ionic strength condition of 1.5 M NaCl (Fig. 4). In the present study, we have functionally and structurally characterized human PAG as a SFK negative regulator. Currently, it is insucient to understand the functions of SFKs in cardiac infarction and insuciency as well as the identication of potential modication sites of PAG and PAG-catalytic kinases from insect. Interestingly, previous

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