Structural Elements Necessary for Oligomerization, Trafficking, and Cell Sorting Function of Paraxial Protocadherin
2007; Elsevier BV; Volume: 282; Issue: 44 Linguagem: Inglês
10.1074/jbc.m705337200
ISSN1083-351X
AutoresXuejun Chen, Caitlyn Molino, Li Liu, Barry M. Gumbiner,
Tópico(s)Skin and Cellular Biology Research
ResumoProtocadherins have been shown to regulate cell adhesion, cell migration, cell survival, and tissue morphogenesis in the embryo and the central nervous system, but little is known about the mechanism of protocadherin function. We previously showed that Xenopus paraxial protocadherin (PAPC) mediates cell sorting and morphogenesis by down-regulating the adhesion activity of a classical cadherin, C-cadherin. Classical cadherins function by forming lateral dimers that are necessary for their adhesive function. However, it is not known whether oligomerization also plays a role in protocadherin function. We show here that PAPC forms oligomers that are stabilized by disulfide bonds formed between conserved Cys residues in the extracellular domain. Disruption of these disulfide bonds by dithiothreitol or mutation of the conserved cysteines results in defects in oligomerization, post-translational modification, trafficking to the cell surface and cell sorting function of PAPC. Furthermore, none of the residues in the cytoplasmic domain of PAPC is required for its cell sorting activity, whereas both the transmembrane domain and the extracellular domain are necessary. Therefore, protein oligomerization and/or protein interactions via the extracellular and transmembrane domains of PAPC are required for its cell sorting function. Protocadherins have been shown to regulate cell adhesion, cell migration, cell survival, and tissue morphogenesis in the embryo and the central nervous system, but little is known about the mechanism of protocadherin function. We previously showed that Xenopus paraxial protocadherin (PAPC) mediates cell sorting and morphogenesis by down-regulating the adhesion activity of a classical cadherin, C-cadherin. Classical cadherins function by forming lateral dimers that are necessary for their adhesive function. However, it is not known whether oligomerization also plays a role in protocadherin function. We show here that PAPC forms oligomers that are stabilized by disulfide bonds formed between conserved Cys residues in the extracellular domain. Disruption of these disulfide bonds by dithiothreitol or mutation of the conserved cysteines results in defects in oligomerization, post-translational modification, trafficking to the cell surface and cell sorting function of PAPC. Furthermore, none of the residues in the cytoplasmic domain of PAPC is required for its cell sorting activity, whereas both the transmembrane domain and the extracellular domain are necessary. Therefore, protein oligomerization and/or protein interactions via the extracellular and transmembrane domains of PAPC are required for its cell sorting function. Protocadherins are type I transmembrane glycoproteins belonging to the cadherin superfamily, encoded by both clustered (protocadherin α, β, and γ) and non-clustered protocadherin genes (1Angst B.D. Kim C. Magee A.I. J. Cell Sci. 2001; 114: 629-641Crossref PubMed Google Scholar, 2Suzuki S.T. J. Cell Sci. 1996; 109: 2609-2611Crossref PubMed Google Scholar, 3Junghans D. Kim I.G. Kemler R. Curr Opin Cell Biol. 2005; 17: 446-452Crossref PubMed Scopus (101) Google Scholar, 4Halbleib J.M. Kim W.J. Genes Dev. 2006; 20: 3199-3214Crossref PubMed Scopus (802) Google Scholar). Their extracellular domains consist of six or seven conserved cadherin repeats, and their cytoplasmic domains are different from that of classical cadherins because they lack the catenin-binding motifs (2Suzuki S.T. J. Cell Sci. 1996; 109: 2609-2611Crossref PubMed Google Scholar). Protocadherins all share high sequence similarity in their extracellular domains, whereas the cytoplasmic domains differ greatly among different subclasses. Limited studies have suggested that protocadherins may function in embryonic development and in the development of the central nervous system (3Junghans D. Kim I.G. Kemler R. Curr Opin Cell Biol. 2005; 17: 446-452Crossref PubMed Scopus (101) Google Scholar, 4Halbleib J.M. Kim W.J. Genes Dev. 2006; 20: 3199-3214Crossref PubMed Scopus (802) Google Scholar). In Xenopus embryos, axial protocadherin (AXPC), paraxial protocadherin (PAPC), 2The abbreviations used are: PAPC, paraxial protocadherin; IL, interleukin; DTT, dithiothreitol; GPI, glycosylphosphatidylinositol; IAA, iodoacetamide; EC, extracellular domain; WT, wild type; β-ME, β-mercaptoethanol. and neural fold protocadherin (NFPC) have been shown to play roles in cell sorting, cell adhesion, and morphogenesis in notochord, paraxial mesoderm, and neural tube, respectively (5Kuroda H. Kim M. Sugimoto K. Hayata T. Asashima M. Dev. Biol. 2002; 244: 267-277Crossref PubMed Scopus (51) Google Scholar, 6Kim S.H. Kim A. Bouwmeester T. Agius E. Robertis E.M. Development. 1998; 125: 4681-4690Crossref PubMed Google Scholar, 7Kim S.H. Kim W.C. De Robertis E.M. Kintner C. Curr. Biol. 2000; 10: 821-830Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar, 9Bradley R.S. Kim A. Kintner C. Curr. Biol. 1998; 8: 325-334Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 10Heggem M.A. Kim R.S. Dev. Cell. 2003; 4: 419-429Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Rashid D. Kim K. Shama L. Bradley R. Dev. Biol. 2006; 291: 170-181Crossref PubMed Scopus (29) Google Scholar). In mouse brain, protocadherin α proteins have been proposed to act as reelin receptors and influence layering and positioning of neurons (12Senzaki K. Kim M. Yagi T. Cell. 1999; 99: 635-647Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Deletion of the protocadherin γ subfamily members lead to cell death of spinal interneurons and neonatal death of mutant mice (13Wang X. Kim J.A. Levi S. Craig A.M. Bradley A. Sanes J.R. Neuron. 2002; 36: 843-854Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Despite these findings, the specific mechanisms of protocadherin function are not yet understood. In contrast to the structures of classical cadherins, which have been well characterized (14Nagar B. Kim M. Ikura M. Rini J.M. Nature. 1996; 380: 360-364Crossref PubMed Scopus (565) Google Scholar, 15Tamura K. Kim W.S. Hendrickson W.A. Colman D.R. Shapiro L. Neuron. 1998; 20: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 16Pertz O. Kim D. Koch A.W. Fauser C. Brancaccio A. Engel J. EMBO J. 1999; 18: 1738-1747Crossref PubMed Scopus (344) Google Scholar, 17Boggon T.J. Kim J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (548) Google Scholar, 18Patel S.D. Kim C. Chen C.P. Bahna F. Rajebhosale M. Arkus N. Schieren I. Jessell T.M. Honig B. Price S.R. Shapiro L. Cell. 2006; 124: 1255-1268Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), very little is known about the structure of protocadherins. An NMR structure of the first ectodomain (EC1), one of a total of 6 EC domains, of protocadherin α4 was solved recently (19Morishita H. Kim M. Murata Y. Shibata N. Udaka K. Higuchi Y. Akutsu H. Yamaguchi T. Yagi T. Ikegami T. J. Biol. Chem. 2006; 281: 33650-33663Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The overall topology of this protocadherin ectodomain is very similar to that of classical cadherins, which is a β sandwich composed of 7 β strands. Classical cadherins are known to function as cell-cell adhesion molecules and they form lateral dimers that are necessary for their adhesive function (20Brieher W.M. Kim A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar). No study has yet been done to determine whether protocadherins also oligomerize and whether oligomerization also plays a role in protocadherin function. The dimerization and adhesive function of classical cadherins requires the conserved Trp2 residue in their EC1 domains (21Gumbiner B.M. Nat. Rev. Mol. Cell Biol. 2005; 6: 622-634Crossref PubMed Scopus (1223) Google Scholar). In a classical cadherin dimer, the Trp2 from each monomer is inserted reciprocally into a hydrophobic pocket of the other molecule. Protocadherins do not contain the Trp2 residue. Instead, nearly all protocadherins contain a highly conserved Tyr residue near the N terminus of the mature protein. Whether this Tyr can mediate dimerization like the Trp2 in classical cadherins is unknown. Furthermore, protocadherins contain numerous conserved cysteines in the EC domains, which could potentially mediate oligomerization by forming intermolecular disulfide bonds. Studies on protocadherin subunit interactions and oligomerization will help us understand the molecular mechanisms governing protocadherin function. Xenopus paraxial protocadherin (PAPC) is specifically expressed in paraxial mesoderm in early frog embryos and involved in the morphogenesis of gastrula and somites (6Kim S.H. Kim A. Bouwmeester T. Agius E. Robertis E.M. Development. 1998; 125: 4681-4690Crossref PubMed Google Scholar, 7Kim S.H. Kim W.C. De Robertis E.M. Kintner C. Curr. Biol. 2000; 10: 821-830Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The extracellular domain of PAPC (PAPC-EC) has six cadherin repeats and shares high sequence homology to all other protocadherins, whereas the cytoplasmic domain of PAPC is much less conserved with only a segment of ∼25 amino acid residues being conserved across species (supplemental Fig. S3). The closest homolog of PAPC in human or mouse is protocadherin 8 (Pcdh8), although the Xenopus ortholog of Pcdh8 is probably a different protocadherin (GenBank™ accession number: AAH74360). PAPC has been shown to mediate cell sorting and morphogenesis in early embryos (6Kim S.H. Kim A. Bouwmeester T. Agius E. Robertis E.M. Development. 1998; 125: 4681-4690Crossref PubMed Google Scholar, 7Kim S.H. Kim W.C. De Robertis E.M. Kintner C. Curr. Biol. 2000; 10: 821-830Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Ectopic expression of PAPC in a small area of Xenopus embryos leads to sorting out of the PAPC-expressing cells from the non-expressing cells and formation of a sharp boundary between them (6Kim S.H. Kim A. Bouwmeester T. Agius E. Robertis E.M. Development. 1998; 125: 4681-4690Crossref PubMed Google Scholar, 8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). We previously showed that PAPC mediates these functions not by directly acting as a homophilic adhesion molecule but by down-regulating the adhesion activity of a classical cadherin, C-cadherin (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). Consistent with this finding, recent evidence suggested that another protocadherin, mouse protocadherin α4, does not mediate homophilic adhesion either (19Morishita H. Kim M. Murata Y. Shibata N. Udaka K. Higuchi Y. Akutsu H. Yamaguchi T. Yagi T. Ikegami T. J. Biol. Chem. 2006; 281: 33650-33663Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Mutoh T. Kim S. Senzaki K. Murata Y. Yagi T. Exp. Cell Res. 2004; 294: 494-508Crossref PubMed Scopus (43) Google Scholar). The roles of oligomerization, disulfide bond formation, conserved Cys and Tyr residues, and different domains in the trafficking and function of PAPC have been analyzed. Given the high conservation of the extracellular domains among different protocadherins, the findings with PAPC likely reflect the properties of other protocadherins as well. PAPC Constructs—Plasmids pCS2+/FL-PAPC(-UTR), pCS2+/M-PAPC, pEE14/PAPC-EC·His have been described (6Kim S.H. Kim A. Bouwmeester T. Agius E. Robertis E.M. Development. 1998; 125: 4681-4690Crossref PubMed Google Scholar, 8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). PAPC constructs in pCS2+ vectors were used for the transfection of CHO cells and the in vitro preparation of capped mRNAs for expression in Xenopus embryos. The pEE14/PAPC-EC·His was constructed for eukaryotic expression of secreted PAPC-EC·His proteins. The complete deletion of the cytoplasmic domain of PAPC was achieved by digesting pCS2+/FL-PAPC with XcmI and XhoI, blunting and re-ligating the ends. The resulting construct, pCS2+/ECTM, contains only the extracellular and transmembrane domains of PAPC (1–711 amino acids) and a short peptide tag resulting from the vector sequence (RASRTIVSRIT*). To construct the glycosylphosphatidylinositol (GPI)-linked PAPC (pCS2+/PAPC-GPI), the extracellular domain of PAPC was fused to the GPI signal sequence of the human decay-accelerating factor by two rounds of PCR and subsequent cloning into the EcoRI-XbaI site of pCS2+. The primers for the PCRs are: SP6 and PAPC-GPI.r (5′-gga tcg atc tgg tgc tgc tct tct gca gat ggt tg-3′) for amplification of PAPC extracellular domain from pCS2+/M-PAPC, and PAPC-GPI.f (5′-gag cag cac cag atc gat cca aat aaa gga agt gga-3′) and GPI3′-XbaI (5′-ctc tag act aag tca gca agc cca t-3′) for amplification of the GPI-anchor signal from decay-accelerating factor in pCB6/Aenv-GPI (a kind gift from Dr. Judy White, University of Virginia). By similar strategy, the fusion of the PAPC extracellular domain with interleukin 2 receptor α (IL2Rα) transmembrane domain (PAPC-IL2R™), the fusion of IL2R extracellular domain with PAPC transmembrane and cytoplasmic domains (IL2R-PAPCTMC), and the point mutation of the M-PAPC transmembrane Cys to Ala (M.C703A) were obtained by PCR methods using pCS2+/FL-PAPC (-UTR), pCS2+/IL2Rα, and/or pCS2+/M-PAPC as primary templates. The coding sequences of resulting fusion or mutant proteins were then inserted into the EcoRI-XbaI or HindIII-XbaI (for IL2R-PAPCTMC) site of pCS2+. The primers used for building these constructs are: SP6, PAPC-IL2R™.f (5′-cag cac cag atc gat tac cag gta gca gtg-3′), PAPC-IL2R™.r (5′-cac tgc tac ctg gta atc gat ctg gtg ctg-3′), IL2R-PAPCTMC.f (5′-tcc aga ttt aca aca gag atg tcc att ata ttc att-3′), IL2R-PAPCTMC.r (5′-aat gaa tat aat gga cat ctc tgt tgt aaa tct gga-3′), C703A.f (5′-gct ggt ggt gct gct ttg cta-3′), C703A.r (5′-tag caa agc agc acc acc agc-3′), and T7. Sp6 and T7 were used for the 2nd round PCR in each case. Site-directed point mutagenesis (Y27S, C90S, C96S, C330S, and C389S) was performed using the QuickChange®-XL Site-Directed Mutagenesis kit (Stratagene) on pEE14/PAPC-EC·His, pCS2+/FL-PAPC(-UTR), or pCS2+/M-PAPC. Double, triple, or quadruple point mutations were achieved by restriction/subcloning approaches or repeated site-directed mutagenesis. The primers for the point mutagenesis are: Y27S.f (5′-att gcc cag tcc tac ata gat gaa-3′), Y27S.r (5′-ttc atc tat gta gga ctg ggc aat-3′), C90S.f (5′-cgg gag cag atc agc agg cag tcc ct-3′), C90S.r (5′-agg gac tgc ctg ctg atc tgc tcc cg-3′), C96S.f (5′-ggc agt ccc ttc aca gca acc tgg ctt tgg-3′), C96S.r (5′-cca aag cca ggt tgc tgt gaa ggg act gcc-3′), C330S.f (5′-caa ccc act gac tgc tac tag taa agt aac tgt tca tat act-3′), C330S.r (5′-agt ata tga aca gtt act tta cta gta gca gtc agt ggg ttg-3′), C389S.f (5′-tct aat gga caa gtt cgc agt act ctt tat gga cat gag-3′), and C389S.r (5′-ctc atg tcc ata aag agt act gcg aac ttg tcc att aga-3′). Cell Lines and Antibodies—CHO cell lines stably expressing FL-PAPC or secreted PAPC-EC·His have been described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). Transient transfection of CHO cells was performed with Lipofectamine 2000 (Invitrogene) in 24-well plates according to the manufacturer's instructions. 0.8 μg of DNA was used for each transfection. Anti-PAPC extracellular domain mAbs, 11A6 and 28F12, were used for immunoblotting of PAPC proteins and have been described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). Anti-IL2R mAb B-B10 (BioSource/Invitrogen Corp) was used for the immunoblot of IL2R-PAPCTMC. Biochemical Analysis of PAPC-EC·His—The preparation and purification of PAPC-EC·His from conditioned media of transfected CHO cells have been described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). For gel filtration analysis, 100 μl of 0.4 mg/ml purified PAPC-EC·His was either treated with 10 mm DTT for 30 min and with 20 mm iodoacetamide for 15 min at room temperature or mock-treated. After centrifugation at 14,000 × g for 10 min, the protein sample was applied to a Superose 12 column (Amersham Biosciences BioSicences/GE Corp), and eluted with 20 mm Tris-HCl, 100 mm NaCl, 1 mm CaCl2, pH7.5. 0.5 ml fractions were collected. For non-reducing SDS-PAGE, 10 μl of each elution fraction was resolved on a 7% gel without DTT treatment. Proteins were then transferred to nitrocellulose filter and subjected to immunoblotting with anti-PAPC mAbs. Typically, the disulfide bond-mediated oligomerization of PAPC-EC·His can be detected by running non-reducing SDS-PAGE and anti-PAPC immunoblotting. When specifically indicated, 1 mm CuCl2 was added for enhanced cross-linking. For chemical cross-linking, purified PAPC-EC·His (50 μg/ml) was dialyzed in 20 mm Hepes, 150 mm NaCl, 1 mm CaCl2, pH 7.4 and then incubated with 1 mm ethylene glycol bis[succinimidylsuccinate] (EGS, from Pierce) at room temperature for 30 min. The cross-linking was quenched by adding glycine, pH7.8 to a final concentration of 0.1 m, and the product was examined by reducing SDS-PAGE and immunoblotting. Biochemical Analysis of Cellular PAPC—Trypsinization and biotinylation assays for assessing PAPC cell surface expression have been described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). The analysis of N-linked glycosylation of PAPC was performed with Endo Hf and peptide N-glycosidase F (PNGase F) (New England BioLabs, Inc.) following the manufacturer's instructions. Briefly, confluent CHO cells expressing PAPC in a 10-cm dish were lysed in 0.5 ml of phosphate-buffered saline containing 1% Nonidet P-40 and protease inhibitor mixture (Roche Applied Science). The lysate was cleared by centrifugation and denatured by adding 1/10 volume of 10× glycoprotein denaturing buffer (New England BioLabs, Inc.) and heating at 100 °C for 5 min. The denatured lysate was then supplemented with appropriate 10× reaction buffers (G5 buffer for Endo Hf; G7 buffer and 10% Nonidet P-40 for PNGase F) provided by the manufacturer to obtain 1× reaction mix. For each 30-μl reaction mix, 1 μl of Endo Hf (1,000 NEB units), PNGase F (500 NEB units), or H2O (as control) was added, and the reactions were incubated at 37 °C for overnight. One-third of each reaction was analyzed by SDS-PAGE and immunoblotting against PAPC. Immunofluorescence Staining of Surface and Total PAPC in Transfected CHO Cells—Transiently transfected CHO cells were incubated with anti-PAPC mAb 28F12 (5 μg/ml) at 4 °C for 45 min, washed 4× with PBS, fixed in -20 °C methanol, blocked with 3% bovine serum albumin in PBS, and labeled with Alexa488-conjugated goat anti-mouse IgG (Invitrogen). After extensive wash, the cells were re-stained with 28F12 and Alexa564-conjugated goat anti-mouse IgG for total PAPC both inside and on the surface of the cells. Immunofluorescence microscopy was performed on a Zeiss Axioplan2 microscope with a Zeiss Neoplan10x objective lens. Images were acquired with a Hamamamstu C4742-95 digital cameral and the Open-lab 4.0 (Improvision) software. Assays using Xenopus Embryos—The preparation, handling, and staging of Xenopus laevis embryos and the procedure for ectopic expression of PAPC or PAPC mutant proteins in Xenopus embryos have been described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). PAPC or PAPC mutant mRNA (1–1.5 ng) was microinjected into the animal hemisphere of 4-cell stage embryos. At stage 9, proteins were extracted from the embryos and subject to anti-PAPC immunoblotting. The cell dispersal assay was done as previously described (8Chen X. Kim B.M. J. Cell Biol. 2006; 174: 301-313Crossref PubMed Scopus (119) Google Scholar). The mRNA (600 pg) of PAPC or PAPC mutants was co-injected with the mRNA of nuclear-localized GFP (200 pg, as lineage tracer) into one blastomere of 32-cell stage embryos. At stage 13–14, the dispersal of the GFP-positive cells was scored under fluorescence microscope. PAPC Forms Disulfide Bond-dependent Homo-oligomers—To better study the properties of the PAPC extracellular domain, a His6-tagged secreted form of the extracellular domain of PAPC (PAPC-EC·His) was purified from conditioned CHO cell media (Fig. 1 and supplemental Fig. S1). We first asked whether PAPC-EC·His forms oligomers as classical cadherins form dimers (20Brieher W.M. Kim A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar). Because the PAPC extracellular domain contains conserved Cys residues, we examined whether PAPC-EC·His forms disulfide bond-linked oligomers. Under reducing conditions, purified PAPC-EC·His migrates at ∼100 kDa on SDS-PAGE gels; whereas under non-reducing conditions, nearly half of the PAPC-EC·His migrates at ∼300 kDa, suggesting that PAPC-EC·His forms disulfide bond-linked homo-oligomers (Fig. 2A). Incubation of PAPC-EC·His with 1 mm of CuCl2, an oxidizer that promotes disulfide bond formation, led to more PAPC-EC·His in the oligomeric form in the non-reducing SDS-PAGE gel (Fig. 2A, lane 3). Moreover, the formation of PAPC-EC·His oligomers can also be detected by chemical cross-linking via EGS, even when subsequently analyzed under reducing conditions (Fig. 2A, lane 4). Compared with the disulfide-cross-linked samples (Fig. 2A, lanes 2 and 3), which still contained a significant portion of monomeric PAPC-EC·His, EGS treated PAPC-EC·His consisted mostly of the higher molecular weight form (Fig. 2A, lane 4). This suggests that some of PAPC-EC·His oligomers are not linked by disulfide bonds.FIGURE 2PAPC forms disulfide bond-dependent oligomers. A, purified PAPC-EC·His forms oligomers that can be covalently linked by disulfide bonds or chemically cross-linked with EGS. Purified PAPC-EC·His was pretreated as indicated, resolved on a non-reducing or reducing (+β-ME) SDS-PAGE gel, and analyzed by anti-PAPC immunoblotting. EGS, ethylene glycol bis[succinimidylsuccinate]. B and C, gel filtration analysis of untreated (B) or DTT/IAA-pretreated (C) PAPC-EC·His. Fractions were analyzed by non-reducing SDS-PAGE and anti-PAPC immunoblotting. Arrows indicate the elution fractions of corresponding molecular weight standards. D, M-PAPC and FL-PAPC form disulfide bond-linked oligomers in transfected CHO cells. Cell lysates of CHO cells expressing either M-PAPC or FL-PAPC were resolved by SDS-PAGE in the presence or absence of DTT, and immunoblotted with anti-PAPC mAb. E, M-PAPC expressed in Xenopus embryos forms oligomers. Lysates of mock-injected control embryos (Con) or M-PAPC-RNA-injected embryos (M) were resolved by SDS-PAGE in the presence or absence of DTT, and blotted with anti-PAPC antibody. F, endogenous PAPC oligomerizes in Xenopus embryos. Lysates of stage 12 embryos were treated with or without DTT and analyzed by non-reducing SDS-PAGE and anti-PAPC immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further characterize the PAPC oligomers and examine the role of disulfide bonds in PAPC oligomerization, we performed gel filtration analysis of purified PAPC-EC·His protein that was either untreated or reduced with DTT and treated with iodoacetamide (IAA) to prevent the re-formation of disulfide bonds (Fig. 2, B and C). Fractions were analyzed by non-reducing SDS-PAGE and immunoblotting against PAPC. Without DTT treatment, purified PAPC-EC·His elutes largely in two pools: one with a peak in fractions 20–21, which corresponds to a size of ∼300 kDa; the other with a peak in fractions 24–25, which corresponds to a size of ∼100 kDa (Fig. 2B). A large portion of the PAPC in the high molecular weight (M.W.) column fractions migrates at ∼300 kDa in a non-reducing gel, whereas most PAPC in the low M.W. fractions migrates at the monomer size of PAPC-EC·His. These results indicate that a significant portion of PAPC-EC·His forms homo-oligomers in solution. Noticeably, a significant portion of PAPC-EC·His in the high M.W. gel filtration peak still runs at the monomer size in non-reducing SDS-PAGE, suggesting that not all PAPC molecules are covalently linked to each other in the oligomer complex and that oligomerization can occur without intermolecular disulfide linkage (Fig. 2B). This is consistent with the cross-linking data shown in Fig. 2A. Although the intermolecular disulfide linkage is not necessary for PAPC-EC·His oligomerization, some intramolecular disulfide bonds must be required, because disruption of all disulfide bonds by DTT and IAA treatment eliminated both the ∼300-kDa band in the non-reducing SDS-PAGE gel and the high M.W. gel filtration peak of PAPC-EC·His (Fig. 2C). These results suggest that the disulfide bonds of PAPC are not only responsible for intermolecular linkage, but are probably also required for maintaining or stabilizing correct PAPC conformation necessary for oligomerization. Therefore, PAPC-EC·His forms oligomers that can exist with or without intermolecular disulfide linkages. Based on the size estimated from both the SDS-PAGE and gel filtration profiles, the size of the PAPC oligomer is consistent with being a homotrimer, although other oligomerization states are possible. To test whether oligomerization is an intrinsic property of PAPC within the cell, we examined the oligomerization state of a truncated transmembrane form of PAPC (M-PAPC, see Fig. 1) and the full-length PAPC (FL-PAPC, see Fig. 1) in either PAPC-expressing CHO cells or in Xenopus embryos. In PAPC-expressing CHO cells, most of either M-PAPC or FL-PAPC exist as disulfide bond-linked oligomers as shown by non-reducing SDS-PAGE (Fig. 2D) and the size of the oligomers is slightly larger than that of PAPC-EC·His oligomers. In Xenopus embryos, over half of exogenously expressed M-PAPC exists as disulfide bond linked oligomers (Fig. 2E), with a similar size as observed in CHO cells. Endogenous FL-PAPC also exists in oligomers (Fig. 2F), again, about 3 times the size of the reduced monomeric polypeptide. Therefore, PAPC forms disulfide bond-dependent oligomers in cells and embryos. Conserved Cys Residues Are Necessary for PAPC Oligomerization, Proper Glycosylation, Trafficking to the Cell Surface, and Function—To investigate whether the disulfide-dependent oligomerization of PAPC is necessary for PAPC function, we decided to mutate the cysteines in the extracellular domain of PAPC. There are five Cys residues in the extracellular region of PAPC: Cys90 and Cys96 in the EC1 domain, Cys330 in the EC3 domain, Cys389 in the EC4 domain, and Cys564 in the EC5 domain. The first four Cys residues are highly conserved, not only among PAPC homologs across species but also in other protocadherins. The fifth Cys, Cys564, is not conserved at all, even among PAPC homologs (supplemental Figs. S2 and S3). The two Cys residues in the EC1 domain are absolutely conserved in all protocadherins; Cys330 and Cys389 are present in most protocadherins but are somewhat less conserved. In known PAPC homologs, Cys330 is conserved in every species, and Cys389 is conserved in most of them (7/8) (supplemental Fig. S3). We generated single (C90S, C96S, or C330S), double (C90/96S, C90/330S, or C96/330S), triple (3S), and quadruple (4S) Cys → Ser mutations in PAPC-EC·His (Fig. 1). PAPC-EC·His proteins bearing these mutations were expressed in CHO cells for examining oligomerization of the secreted mutant proteins. Surprisingly, all these mutations, except for the C330S single mutation, led to failure of PAPC secretion (compared with the wild-type protein), even though similar amount of mutant proteins were detected in total CHO cell lysates (Fig. 3A). The secreted C330S mutant still makes intermolecular disulfide bonds as shown by non-reducing SDS-PAGE (Fig. 3B), suggesting that mutating Cys330 alone is not enough to prevent disulfide bond formation. We therefore examined the soluble Cys mutant PAPC-EC·His proteins from total cell lysates by non-reducing SDS-PAGE, and found that all mutants, including the quadruple Cys mutant, could still form oligomeric species (Fig. 3C). However, the gel band patterns of mutant oligomers were much more variable than the wild-type PAPC oligomers, with multiple additional bands observed on the gel (Fig. 3C). Notably, the unreduced 4S mutant oligomer migrates at 250 kDa, smaller than the ∼300-kDa wild type oligomer (Fig. 3C). Protein degradation is unlikely the cause of the difference in band M.W., because every PAPC mutant protein ran as a single monomeric band in the reducing SDS-PAGE gel (Fig. 3A), without any lower M.W. degradation product observed (data not shown). The fact that the mutant oligomers, except for the C330S, cannot be secreted (Fig. 3A) suggests that they are different from the wild type ones. Therefore, we conclude that mutation of the conserved Cys residues in soluble PAPC-EC·His results in abnormal protein oligomerization and failure of protein secretion. Because membrane association could limit the orientation of PAPC molecules and prevent the non-physiological oligomerization observed in soluble PAPC-EC·His Cys mutants, we decided to determine the roles of Cys residues in PAPC oligomerization using the transmembrane forms of PAPC, either FL-PAPC or M-PAPC (Fig. 1). We generated single (FL.C96S), triple (M.3S) and quadruple (M.4S) Cys mutants (Fig. 1), which represent various Cys mutants that exhibited different oligomerization patterns in PAPC-EC·His (Fig. 3C
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