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• W2159410930 abstract "Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53. Consensus profiles were established to screen data bases for novel animal L-type lectins. The profiles were generated from linear sequence motifs of the human L-type lectin-like membrane proteins ERGIC-53, ERGL, and VIP36 and by optimal alignment of the entire carbohydrate recognition domain of these proteins. The search revealed numerous orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast, as well as the sequence of a novel family member related to VIP36, named VIPL for VIP36-like. Sequence analysis suggests that VIPL is a ubiquitously expressed protein and appeared earlier in evolution than VIP36. The cDNA of VIPL was cloned and expressed in cell culture. VIPL is a high-mannose type I membrane glycoprotein with similar domain organization as VIP36. Unlike VIP36 and ERGIC-53 that are predominantly associated with postendoplasmic reticulum (ER) membranes and cycle in the early secretory pathway, VIPL is a non-cycling resident protein of the ER. Mutagenesis experiments indicate that ER retention of VIPL involves a RKR di-arginine signal. Overexpression of VIPL redistributed ERGIC-53 to the ER without affecting the cycling of the KDEL-receptor and the overall morphology of the early secretory pathway. The results suggest that VIPL may function as a regulator of ERGIC-53. endoplasmic reticulum carbohydrate recognition domain hemagglutinin brefeldin A ER-Golgi intermediate compartment VIP36-like L-type lectin-like domain of animal lectins Understanding the molecular basis of secretion requires knowledge of how secretory proteins are correctly folded and assembled in a process known as quality control, and how these itinerant proteins are sorted from resident proteins along the secretory pathway. Evidence is mounting that intracellular lectins play an important role in these processes (1Hauri H. Appenzeller C. Kuhn F. Nufer O. FEBS Lett. 2000; 476: 32-37Crossref PubMed Scopus (131) Google Scholar, 2Ellgaard L. Helenius A. Curr. Opin. Cell. Biol. 2001; 13: 431-437Crossref PubMed Scopus (333) Google Scholar). A majority of secretory proteins acquiresN-glycans during translocation into the ER,1 and these modifications are most often required for the folding of the proteins. The folding process is assisted by the membrane lectin calnexin and the related soluble lectin calreticulin. These two lectins recognize monoglucosylated trimming intermediates and, in a remarkable cycle of de- and reglucosylation, guarantee that only correctly folded glycoproteins leave the ER (2Ellgaard L. Helenius A. Curr. Opin. Cell. Biol. 2001; 13: 431-437Crossref PubMed Scopus (333) Google Scholar, 3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Chevet E. Cameron P.H. Pelletier M.F. Thomas D.Y. Bergeron J.J. Curr. Opin. Struct. Biol. 2001; 11: 120-124Crossref PubMed Scopus (106) Google Scholar). If folding is unsuccessful, newly synthesized glycoproteins are degraded by the proteasome after retrotranslocation to the cytosol. Degradation appears to involve calnexin and an ER α-mannosidase-like protein, termed EDEM in mammalian cells, that enhances degradation of misfolded glycoproteins carrying Man8(GlcNAc)2glycans (5Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 6Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar, 7Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar). The strict functional dependence of EDEM on a Man8(GlcNAc)2 structure suggests it may function as a lectin. Correctly folded glycoproteins leave the ER in COPII-coated vesicles (8Antonny B. Schekman R. Curr. Opin. Cell Biol. 2001; 13: 438-443Crossref PubMed Scopus (159) Google Scholar) and are transported to the Golgi via the ER-Golgi intermediate compartment (ERGIC, Ref. 9Hauri H.P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar). The exit signals that direct proteins out of the ER are poorly understood with few exceptions (10Nufer O. Guldbrandsen S. Degen M. Kappeler F. Paccaud J.P. Tani K. Hauri H.P. J. Cell Sci. 2002; 115: 619-628Crossref PubMed Google Scholar). The mannose lectin ERGIC-53 (p58 in rat) mediates efficient ER-exit of some secretory glycoproteins by serving as a transport receptor (9Hauri H.P. Kappeler F. Andersson H. Appenzeller C. J. Cell Sci. 2000; 113: 587-596Crossref PubMed Google Scholar, 11Arar C. Carpentier V. Le Caer J.P. Monsigny M. Legrand A. Roche A.C. J. Biol. Chem. 1995; 270: 3551-3553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 12Itin C. Roche A.C. Monsigny M. Hauri H.P. Mol. Biol. Cell. 1996; 7: 483-493Crossref PubMed Scopus (154) Google Scholar, 13Lahtinen U. Hellman U. Wernstedt C. Saraste J. Pettersson R.F. J. Biol. Chem. 1996; 271: 4031-4037Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). ERGIC-53-assisted proteins include blood coagulation factors V and VIII (14Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Wheatley M.A. Moussalli M.J. Hauri H.P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar), cathepsin C (15Vollenweider F. Kappeler F. Itin C. Hauri H.P. J. Cell Biol. 1998; 142: 377-389Crossref PubMed Scopus (133) Google Scholar), and cathepsin Z (16Appenzeller C. Andersson H. Kappeler F. Hauri H.P. Nat. Cell Biol. 1999; 1: 330-334Crossref PubMed Scopus (269) Google Scholar). ERGIC-53 is a major membrane protein of the tubulovesicular clusters of the ERGIC (17Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (374) Google Scholar) and efficiently cycles between ERGIC and ER (18Lippincott-Schwartz J. Donaldson J.G. Schweizer A. Berger E.G. Hauri H.P. Yuan L.C. Klausner R.D. Cell. 1990; 60: 821-836Abstract Full Text PDF PubMed Scopus (729) Google Scholar, 19Aridor M. Bannykh S.I. Rowe T. Balch W.E. J. Cell Biol. 1995; 131: 875-893Crossref PubMed Scopus (340) Google Scholar, 20Klumperman J. Schweizer A. Clausen H. Tang B.L. Hong W. Oorschot V. Hauri H.P. J. Cell Sci. 1998; 111: 3411-3425PubMed Google Scholar). A second lectin cycling in the early secretory pathway, VIP36 is related to ERGIC-53 (21Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (131) Google Scholar). VIP36 is associated with ERGIC and Golgi (22Fullekrug J. Scheiffele P. Simons K. J. Cell Sci. 1999; 112: 2813-2821Crossref PubMed Google Scholar) and to some extent with the plasma membrane (23Yamashita K. Hara-Kuge S. Ohkura T. Biochim. Biophys. Acta. 1999; 1473: 147-160Crossref PubMed Scopus (47) Google Scholar). VIP36 binds mannose (24Hara-Kuge S. Ohkura T. Seko A. Yamashita K. Glycobiology. 1999; 9: 833-839Crossref PubMed Scopus (69) Google Scholar) and GalNAc (25Fiedler K. Simons K. J. Cell Sci. 1996; 109: 271-276Crossref PubMed Google Scholar) and may operate in glycoprotein transport to the apical plasma membrane of polarized cells (26Hara-Kuge S. Ohkura T. Ideo H. Shimada O. Atsumi S. Yamashita K. J. Biol. Chem. 2002; 277: 16332-16339Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). ERGIC-53 and VIP36 are type I membrane proteins. In their luminal segment they carry a single domain of about 200 amino acids that exhibits sequence similarity to the carbohydrate recognition domain (CRD) of soluble lectins of leguminous plants (21Fiedler K. Simons K. Cell. 1994; 77: 625-626Abstract Full Text PDF PubMed Scopus (131) Google Scholar). This domain is known as L-type CRD (27Dodd R.B. Drickamer K. Glycobiology. 2001; 11: 71R-79RCrossref PubMed Scopus (321) Google Scholar). The L-type lectin-like domain of animal lectins is designated LTLD in the present study. The family of plant L-type lectins includes numerous members whereas only three L-type lectins are known in animals: ERGIC-53, VIP36, and a recently discovered ERGIC-53-like protein termed ERGL (28Yerushalmi N. Keppler-Hafkemeyer A. Vasmatzis G. Liu X.F. Olsson P. Bera T.K. Duray P. Lee B. Pastan I. Gene (Amst.). 2001; 265: 55-60Crossref PubMed Scopus (27) Google Scholar). Unlike ERGIC-53 and VIP36 that are ubiquitously expressed proteins, the ERGLgene is expressed in a limited number of tissues only, with highest mRNA levels in normal and neoplastic prostate cells. The ERGL protein remains to be characterized. We wondered if there are additional, yet unidentified members of the animal L-type lectin family. To identify such proteins we established profiles characteristic for animal L-type lectins. By scanning data bases with these profiles we identified orthologous and homologous L-type lectin-like proteins in animals, protozoans, and yeast. A novel member of this family displays molecular similarities with VIP36 and was therefore dubbed VIPL (for VIP36-like). The corresponding protein was cloned and expressed in cell culture. VIPL was found to localize to the ER and to affect the recycling of ERGIC-53. Mouse monoclonal antibodies (mAb): 9E10.2 (IgG1) against c-Myc, 12CA5 (IgG2b) and 16B12 (IgG1, CRP) against the hemagglutin (HA) epitope, A1/182 (IgG2a) against BAP31 (20Klumperman J. Schweizer A. Clausen H. Tang B.L. Hong W. Oorschot V. Hauri H.P. J. Cell Sci. 1998; 111: 3411-3425PubMed Google Scholar), A1/296 (IgG2a) against CLIMP-63 (29Schweizer A. Ericsson M. Bachi T. Griffiths G. Hauri H.P. J. Cell Sci. 1993; 104: 671-683Crossref PubMed Google Scholar), G1/93 (IgG1) against ERGIC-53 (17Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (374) Google Scholar), G1/133 (IgG1) against giantin (30Linstedt A.D. Hauri H.P. Mol. Biol. Cell. 1993; 4: 679-693Crossref PubMed Scopus (355) Google Scholar), A1/118 (IgG1) against GPP130 (31Linstedt A.D. Mehta A. Suhan J. Reggio H. Hauri H.P. Mol. Biol. Cell. 1997; 8: 1073-1087Crossref PubMed Scopus (99) Google Scholar). Rabbit antibodies against human KDEL receptor and rat Sec31p were kindly provided by H.-D. Söling, University of Göttingen, and F. Gorelick, Yale University, respectively. Secondary goat-anti-rabbit and goat-anti-mouse antibodies (either against whole IgG (H+L) or IgG-subtypes) conjugated with AlexaFluor 488 or AlexaFluor 568 were from Molecular Probes (The Netherlands). Secondary peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse antibodies were from Jackson ImmunoResearch Laboratories Inc. Brefeldin A (BFA) was from Epicentre Technologies, and cell culture media and reagents were from Invitrogen and Sigma. Standard molecular biology protocols were adapted from Refs. 32Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current protocols in molecular biology. Wiley, New York1997Google Scholar or 33Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning: A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar. Oligonucleotides were from Microsynth (Switzerland). ERGIC-53 constructs containing a Myc-tag and an artificial N-glycosylation site, termed GM, have been described (34Itin C. Schindler R. Hauri H.P. J. Cell Biol. 1995; 131: 57-67Crossref PubMed Scopus (101) Google Scholar, 35Kappeler F. Klopfenstein D.R. Foguet M. Paccaud J.P. Hauri H.P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). VIPL cDNA was obtained by RT-PCR. Total RNA of subconfluent HepG2 cells was isolated using peqGOLD RNApure reagent (peqLab, Biotech GmbH, Germany) with a protocol modified from Ref. 36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63084) Google Scholar. Reverse transcription was done with 2 μg of total RNA using Omniscript RT enzyme (Qiagen, Switzerland) and oligo d (T)14V primer. Subsequent PCR was performed with VIPL-specific primer pairs and Tgo DNA proof-reading polymerase (Roche Molecular Biochemicals, Switzerland). The 5′-end VIPL primer contained an additional BamHI site and maintains the Kozak transcription initiation sequence preceding the start AUG codon of VIPL. The 3′-end primer had a XbaI site after the stop codon. The resulting cDNA was cloned into pcDNA 3.1 vector (Invitrogen) via BamHI and XbaI sites. A HA epitope (YPYDVPDYA) was introduced downstream of the signal sequence cleavage site between amino acids 44 and 45 of full-length VIPL. This construct was generated by PCR-based splicing (37Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2634) Google Scholar). Selected amino acids were substituted by oligonucleotide-directed PCR mutagenesis or sequence-overlap-extension PCR (38Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6809) Google Scholar), and mutant fragments were recloned via BamHI/XbaI orSacII/XbaI sites into VIPL constructs. For creating chimeric GM constructs with the cytoplasmic tail of VIPL (GM-ViTa), a BglII restriction site was introduced adjacent to the transmembrane domain of GM by silent mutagenesis changing the codon of the arginine 499 in the tail to AGA. This site allows insertion of fragments via BglII and XbaI into GM constructs. A cDNA encoding the cytoplasmic tail of VIPL was prepared by annealing complementary oligonucleotides as described (10Nufer O. Guldbrandsen S. Degen M. Kappeler F. Paccaud J.P. Tani K. Hauri H.P. J. Cell Sci. 2002; 115: 619-628Crossref PubMed Google Scholar) and cloned into the GM construct via BglII andXbaI sites. Additional mutations were introduced by PCR and recloned as AccI/XbaI fragments into GM-ViTa construct. All constructs were confirmed by sequencing using standard methods and an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, Switzerland). COS-1 cells were cultured and transfected (DEAE-dextran method) as described (35Kappeler F. Klopfenstein D.R. Foguet M. Paccaud J.P. Hauri H.P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). HEK293 cells (kindly provided by T. Meier, Myocontract, Switzerland) were cultured in Dulbecco's minimal essential medium (4.5 g/liter glucose) supplemented with 10% fetal calf serum and 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml fungizone. HEK293 cells were transfected by the calcium phosphate precipitation method (32Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current protocols in molecular biology. Wiley, New York1997Google Scholar). HepG2 cells were cultured as described (20Klumperman J. Schweizer A. Clausen H. Tang B.L. Hong W. Oorschot V. Hauri H.P. J. Cell Sci. 1998; 111: 3411-3425PubMed Google Scholar) and transfected by use of FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Switzerland). For immunofluorescence experiments cells were plated in poly-l-lysine-coated 8-well multichamber glass slides (Lab Tek, Nalgene-Nunc Intl.). All cultures were grown at 37 °C with 5% CO2 in humidified air, and transfection of cells was carried out 1 day after plating. 42 h after transfection the cells were washed twice with phosphate-buffered saline, starved in labeling medium (MEM without methionine, supplemented with 10% dialyzed fetal calf serum) and pulsed with 100 μCi/ml [35S]methionine/cysteine (EasyTag™ EXPRE35S35S Protein Labeling, PerkinElmer Life Sciences). Cells were immediately processed or chased with complete Dulbecco's minimal essential medium containing 10 mml-methionine. For immunoprecipitation the cells were washed twice with ice-cold phosphate-buffered saline and resuspended in lysis buffer (100 mm sodium phosphate, 1% Triton X-100, pH 8) supplemented with 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 0.5 μg/ml pepstatin). In some experiments 20 mm iodoacetamide or N-methylmaleimide was included in the lysis buffer. Cells were lysed by passing them five times through a 25-gauge needle. After 1 h on ice, the lysate was cleared by centrifugation at 100,000 × g for 1 h. The supernatant was added to protein A-Sepharose beads (Amersham Biosciences) to which antibodies had been prebound. After incubation for at least 1 h on a rotary shaker in the cold, the beads were washed four times with lysis buffer, once with 100 mmsodium phosphate (pH 8) and once with 10 mm sodium phosphate (pH 8). For digestion with endoglycosidase H (endo H) the immunoprecipitates were boiled for 3 min in 50 mm TrisCl, 1% SDS, and 0.1 mβ-mercaptoethanol (pH 6.8). An equal volume of 0.15 msodium citrate (pH 5.3) supplemented with protease inhibitors was added, and digestion with 10 milliunits of endo H (Roche Molecular Biochemicals) was carried out at 37 °C overnight. For digestion with endoglycosidase F (PNGase F), the immunoisolates were boiled for 3 min in 100 mm sodium phosphate, 0.1% SDS, 0.1 mmβ-mercaptoethanol, and 10 mm EDTA (pH 7.2). An equal volume of the same buffer containing 1% Triton X-100 instead of SDS was added and the sample was incubated with 400 milliunits of PNGase F (Roche Molecular Biochemicals, Switzerland) at 37 °C overnight. Membrane association of proteins was tested by the sodium carbonate procedure (39Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1380) Google Scholar). After metabolic labeling, the cells were collected in ice-cold homogenization buffer (20 mm Hepes/KOH, 300 mm sucrose, 0.2 mm phenylmethylsulfonyl fluoride, 20 mm NEM, pH 7.4), resuspended in 0.1 m sodium carbonate (pH 11.5), passed 10 times through a G25 needle, and kept on ice for 30 min. The sample was then layered onto a small cushion of homogenization buffer, and membranes were pelleted by centrifugation at 100,000 ×g for 1 h and resuspended in lysis buffer (see immunoprecipitation). The pH of the supernatant was neutralized by adding one-fifth volume of 0.5 m potassium phosphate (pH 8), and Triton X-100 was added to 1% final concentration. All samples were kept on ice for 30 min before centrifugation at 100,000 ×g for 1 h. The supernatants were subjected to immunoprecipitation. 42 h after transfection with VIPL-HA cDNA, HepG2 cells were incubated for 3 h with 5 μg/ml BFA or with solvent only before subcellular fractionation using Nycodenz gradients (35Kappeler F. Klopfenstein D.R. Foguet M. Paccaud J.P. Hauri H.P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The distribution of organelle markers and VIPL-HA was determined by Western blotting. Samples were separated by gradient SDS-PAGE. Radioactivity was visualized by fluorography using sodium salicylate and BioMax MR-1 films (Rochester). Fluorograms were quantified with a ChemImagerTM and AlphaEaseTM software (Alpha Inotech Corporation). For Western blotting, proteins were transferred to nitrocellulose B85 (45 μm, Schleicher & Schuell, Germany) at 100 V for 1 h in the cold using transfer buffer containing 15.6 mm Tris, 120 mm glycine, and 20% (v/v) methanol (pH 8.4). The nitrocellulose was rinsed with 50% methanol and stained by Amido Black (Serva, Germany). All subsequent incubations were in PBS containing 5% nonfat dry milk and 0.05% Tween 20 (Serva, Germany): Blocking for 1 h, incubation with the first antibody for 60 min, rinsing three times 10 min, incubation with peroxidase-coupled secondary antibody for 60 min, and rinsing 10 min. After three final 10-min washes with phosphate-buffered saline containing 0.05% Tween 20, the nitrocellulose was processed using enhanced chemiluminescence (ChemiGlow ECL reagent, AlphaInotech Corp.) and exposed to BioMax MR-1 films or directly analyzed in a ChemImagerTM. The procedure has been described (10Nufer O. Guldbrandsen S. Degen M. Kappeler F. Paccaud J.P. Tani K. Hauri H.P. J. Cell Sci. 2002; 115: 619-628Crossref PubMed Google Scholar). Specimens were examined with a Polyvar microscope or a Leica confocal laser scanning microscope. The GCG programs (Madison, WI) were used for sequence analysis (40Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11534) Google Scholar). Other software used is available at ExPASy server (www.expasy.org/) (41Appel R.D. Bairoch A. Hochstrasser D.F. Trends Biochem. Sci. 1994; 19: 258-260Abstract Full Text PDF PubMed Scopus (512) Google Scholar). SwissProt (release 40.7) and TrEMBL/release 19.1) data bases were searched. MEME/MotifSearch (42Bailey T.L. Gribskov M. J. Comput. Biol. 1998; 5: 211-221Crossref PubMed Scopus (164) Google Scholar) was performed with full-length sequences of human ERGIC-53, ERGL, and VIP36 with default settings. The algorithm automatically generated a profile, based on settings with 6 motifs of 8 amino acids in length each with one or zero occurrence in each sequence. A ProfileSearch (43Gribskov M. McLachlan A.D. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4355-4358Crossref PubMed Scopus (1114) Google Scholar) against SwissProt and TrEMBL data bases was done with a profile generated of the alignment of ERGIC-53 (amino acids 44–292), VIP36 (amino acids 50–286), and ERGL (amino acids 31–271). For secondary structure predictions we used the GCG program PeptideStructure and PredictProtein (44Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). Potential sites for domains and motifs were identified by MOTIF search in the Prosite library (45Hofmann K. Bucher P. Falquet L. Bairoch A. Nucleic Acids Res. 1999; 27: 215-219Crossref PubMed Scopus (1004) Google Scholar) and by PSORT (psort.ims.u-tokyo.ac.jp). Coiled coil regions were identified with CoilScan (46Lupas A. Methods Enzymol. 1996; 266: 513-525Crossref PubMed Google Scholar), and signal sequences with SPScan and signalP (47Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Int. J. Neural Syst. 1997; 8: 581-599Crossref PubMed Google Scholar). For detection of putative transmembrane domains the programs HMMTOP (48Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (946) Google Scholar), and TMpred were applied in combination with structure predictions. The phylogenetic tree was constructed using the GrowTree software of the GCG package, and distance correction was calculated according to Jukes-Cantor. Profiles for the identification of lectins have been established in several data bases (27Dodd R.B. Drickamer K. Glycobiology. 2001; 11: 71R-79RCrossref PubMed Scopus (321) Google Scholar). For plant L-type lectins such profiles include PS00307 and PS00308 in PROSITE (45Hofmann K. Bucher P. Falquet L. Bairoch A. Nucleic Acids Res. 1999; 27: 215-219Crossref PubMed Scopus (1004) Google Scholar) and PF00138 and PF00139 in Pfam (49Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2005) Google Scholar). We noticed, however, that these profiles do not identify animal L-type lectins accurately in the existing data bases. Therefore, we developed new consensus profiles specific for animal L-type lectins. In a first approach we used software MEME/MotifSearch of GCG (42Bailey T.L. Gribskov M. J. Comput. Biol. 1998; 5: 211-221Crossref PubMed Scopus (164) Google Scholar) to establish a consensus motif pattern from non-aligned full-length sequences of the known human L-type lectins ERGIC-53, ERGL, and VIP36. The algorithm generated a consensus motif pattern of 6 linear sequence motifs of 8 amino acids each with single occurrence in a defined order. All sequences were restricted to the LTLD. One motif encompassed part of loop A of the CRD of l-type lectins (50Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (190) Google Scholar) that includes a conserved aspartate required for sugar and metal binding (Fig.1 B). Two motifs covered the two cysteines known to form an intracellular disulfide bond in native ERGIC-53 (Ref. 51Velloso L.M. Svensson K. Schneider G. Pettersson R.F. Lindqvist Y. J. Biol. Chem. 2002; 277: 15979-15984Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and our own observations). The cysteines are conserved in the LTLD of all three human lectins and in most of their orthologs assembled in Fig. 1. The other four motifs encompassed conserved regions of β-sheets 6 and 15 of ERGIC-53/p58 as well as the 310 helix turn that separates the β-sheets 1a and 1b of ERGIC-53/p58 (51Velloso L.M. Svensson K. Schneider G. Pettersson R.F. Lindqvist Y. J. Biol. Chem. 2002; 277: 15979-15984Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). A second consensus profile for proteins with a putative LTLD was generated by a ProfileSearch method included in the GCG package (43Gribskov M. McLachlan A.D. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4355-4358Crossref PubMed Scopus (1114) Google Scholar). This approach is based on a sequence matrix profile that was generated on the optimal alignment of the LTLDs of human ERGIC-53, ERGL, and VIP36. The similarity of LTLDs was found to be 58% for ERGIC-53 and ERGL, 46% for ERGIC-53 and VIP36, and 45% for VIP36 and ERGL. The algorithm of the profile scan calculates the score (quality) of the optimal alignment between the consensus profile and each sequence in the scanned data bases. The scored proteins of both scanning methods were then analyzed by alignment and comparison to animal L-type lectins, and the presence of residues functionally important for metal/sugar binding was investigated. Moreover, structural features of the LTLD were examined by hydrophilicity plots and secondary structure predictions. Finally we analyzed the identified proteins for possible protein motifs and domains. Both profile scans recovered all the known orthologs of ERGIC-53, ERGL, and of VIP36 from the data bases (Table I and Fig. 1 A). The scores were higher for ERGIC-53 orthologs since profile characteristics were contributed mostly by ERGIC-53 and ERGL and to a lesser extent by VIP36. Two entries (SpTrEMBL accession numbers Q9H0V9 and Q9BQ14) also appeared with high scores. They stand for the same sequence that encodes a novel VIP36-like protein, we termed VIPL (VIP36-like, see below).Table IOrthologs and homologs of animal L-type lectinsGroupOrganismACC1-aAnnotated SwissProt or NCBI protein entries. SwissProt accession number.Alternative ACC1-b2nd SwissProt acc or NCBI acc.ACC of fragmentsNameERGIC-53ManP49257Q12895Q9UQG1–6ERGIC-53, LMANMonkeyC. aethiopsQ9TU32RatQ62902p58MouseQ9D0F3FrogX. laevisQ91671FlyD. melanogasterQ9V3A8CG6822, RheaWormC. elegansP90913K07A1.8TunicataP. misakiensisQ9GR90Putative mannose-specific lectinSlime moldD. discoideumQ8T2B7Putative chemotaxis proteinYeastS. cerevisiaeP43555Emp47pS. cerevisiaeQ12396Emp46pS. pombeO42707ERGLManQ9HAT1ERGLMouseQ8VCD3Aah20188VIP36ManQ12907gp36bDogP49256VIP36MouseQ9DBH5Q9CXG7FishP. flesusQ98TL0PigQ9XSA8VIPLManQ9H0V9Q9BQ14VIPL1-cThis study.MouseXP_129848FishD. rerioAam34658FlyD. melanogasterQ9VCC2Aam29497CG5510WormC. elegansQ22170T04G9.3YeastS. pombeO94401OtherYeastS. cerevisiaeP36137Uip5ProtozoaT. cruciQ9GPB0Antigen 38L. majorQ9GRK5Lectin-related protein1-a Annotated SwissProt or NCBI protein entries. SwissProt accession number.1-b 2nd SwissProt acc or NCBI acc.1-c This study. Open table in a new tab With lower scores the scans also identified lectin-like proteins in yeast. These proteins are the ERGIC-53 like Emp47p (52Schroder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar) and its close relative Emp46p of Saccharomyces cerevisiae (56% similarity to each other) and two entries of Schizosaccharomyces pombe(SpTrEMBL O42707 and O94401). Emp47p and Emp46p are type I membrane proteins with a putative LTLD fold and a stalk containing a coiled coil domain preceding the transmembrane domain. Emp47p contains a DXL(X)5N metal/sugar binding motif in loop C of the LTLD that is found in the plant lectins UEA I and II as well as LAA I (50Sharma V. Surolia A. J. Mol. Biol. 1997; 267: 433-445Crossref PubMed Scopus (190) Google Scholar). By contrast, animal lectins have a metal/sugar binding site of the DXF/YXN type in loop C that is common to most plant lectins (Fig. 1 B). The Emp46p protein does not contain any of t" @default.
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• W2159410930 title "Profile-based Data Base Scanning for Animal L-type Lectins and Characterization of VIPL, a Novel VIP36-like Endoplasmic Reticulum Protein" @default.
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