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Discriminating Facts from Artefacts in the Secreted Ly-6 Protein Family ,[object Object],[object Object],[object Object]
Outline ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Introduction:  Quirks that Lurk in Databases ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Rat Urine    HPLC    Intact MALDI     N-Terminal  Sequence High-speed microbore column
Rat Urine    2D-Gel    Trypsin    MS/MS    PepSea  Search    EST hits ,[object Object],[object Object],[object Object],[object Object]
EST AA893514 vs. dbEST:  30 Rat Hits at 95% to 100% Identity
Assembly of Rat Urinary Proteins 1 and 2 ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],verified signal peptide     RUP1  MGKHILLLPLGLSLLMSSLLA LQ C FRCTSFDSTGFCHVGRQK C QTYP DEICAWVVVTTRD ||| ||||||||||||||||||||||| |:||||:|:|||: |||||||||||||||||| RUP2  MGKPILLLPLGLSLLMSSLLA LQ C FRCESLDSTGLCRVGRRI C QTYP DEICAWVVVTTRD RU P1  GKFVYG NQS CAECNATTVEHGSLIVSTNCCSATPFCNMVHR  101 ||||||||||||| :|||||||||:|||||||||||||||  RUP2  GKFVYG NQS CAECIGTTVEHGSLIISTNCCSATPFCNMVHP  101
RUP3: Independent MS-based Identification by Wait et al.  “Proteins of rat serum, urine and CSF:VI”  Electrophoresis  22, 3043-3052 (2001) RUP1  MGKPILLLPLGLSLLMSSLLALQCFR CESLDSTGLCRVGR RICQTYPDEICAWVVVTTRD RUP2  MGKHILLLPLGLSLLMSSLLALQCFR CTSFDSTGFCHVGR QKCQTYPDEICAWVVVTTRD RUP3  MGKHILLLPLGLSLLMSSLLALQCFR CISFDSTGFCYVGR HICQTYPDEICAWVVVTTRD *** *********************** * **** * ***. ****************** RUP1  GKFVYGNQSCAECIGTTVEHGSLIISTNCCSATPFCNMVHP EST  AA800439 RUP2  GKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR EST  AA893514   RUP3  GKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR EST  AA893518   *************  *********.***************
RUP Paralogues Define a New Family of Secreted Ly-6 Proteins
A Quirky Result:  Solid Matches Between RUP2 and Four Unrelated mRNAs ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Three RUP-like Chimeras and a Pre-mRNA L07806  F1-ATPase inhibitor AF368860  UTR  F1-ATPase inhib L15618  casein kinase II alpha   J03621  mito succinyl-CoA synthase alpha
Translation Matches for the Chimeras Reveal a Cryptic Protein   RUP-2  28  TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 101 TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 417 TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 196 L07806 Rattus rattus mitochondrial IF1 protein mRNA RUP-2: 59  RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 101 RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 708 RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 580 L15618 Rat casein kinase II alpha subunit (CK2) mRNA RUP-2  24  CFRCTSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMV 99 CF C + +S G C+  C  +P E+CA  V+T +DGKFVYGNQSCAEC+  TVEHGSLIVSTNCCSAT FCN+V 50   CFECGNLNSMGICNFRTAVCYAHPGEVCA-SVLTYKDGKFVYGNQSCAECSGRTVEHGSLIVSTNCCSATSFCNIV   274 J03621 Rat mitochondrial succinyl-CoA synthetase alpha subunit
RUP1 Gene Structure
Matching the Chimeras Against the Rat Genome ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Multiple Loci on Rat Chromosome 8: Erroneous Mapping of the Chimeras L15618  casein kinase II alpha L07806  F1-ATPase inhibitor AF198441 Rat RUP2  AF198442 Rat spleen protein 1
What Caused the Chimeras? ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Protein Database Entries from the Chimera and Pre mRNA   ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
The L07806 Chimera Caused Errors in U niGene
RUP Gene Family on Rat 8q21
Rat and Mouse RUP Homologues are Highly Diverged
Sequences Conserved in Rat but Divergent in Mouse
Homologues in Five Mammals but True Orthology Unclear
Remote Human Homolgues but no Strict  Ortholgues  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Threading Reveals Homology between RUP1,  Lynx1 and Snake Toxin Structures Lynx1, an Endogenous Toxin-like modulator of AChRs in the CNS,
Why so Few Apparent Orthologues?
P55000 : Antineoplastic Urinary Protein/S ecreted Mammalian Ly-6/uPAR Related Protein – Equivocal Annotation
Linking Sequence to Function: the Lost Keyword Problem  (PubMed Queries in red) ,[object Object],[object Object],[object Object]
Mouse Ly-6-like Caltrin:  Sequence Errors,  Unverified Reported Function, New Name and New Function?
Confusion Over Caltrin:  5 Different  Sequences  in SwissProt; 22 PubMed Citations ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Limited Knolwedge for the Short Ly-6 Proteins ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Summary of the Bioinformatic Pitfalls ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Conclusions ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Acknowledgments, Reference and Database Entries  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Human Short Ly6 Proteins None - - 18 + - 11q24.2 113 LVLF31 Genset (sec), USDOH CyC PA2 - 21 + + 11q24.2 126 PATE HGS, ARS, Biovision (partial) Ly6 - 22 + + 8q24.3 103 SLURP1 Genentech, HGS, Incyte Ly6 103 22 + - 8q24.3 125 RGTR43 Genentch (sec/tm) ZymoGenetics Ly6 - 22 + + 8q24.3 97 SLURP2 Curagen, Hyseq, HGS (sec),  Incyte (sec)  Genset (partial) Ly6 91 19 + + 8q24.3 115 LYNX1 Patents InterPro GPI Sigpep ESTs Ens Chrom Size Name
VertebrateShort Ly6 Proteins
Searches Against Rat ESTs Confirmed the Three  mRNAs as Chimeras J03621 L07806 L15618
mRNA Anomaly No. 4:  Unspliced? ,[object Object],[object Object],[object Object],BLAST vs Rat ESTs RUP-4?  MGKHILLLPLVLSLLMSSLLALQCIQCARIDSRGICRHDIYICHADSDEVCSWVVATTRD  MGKHILLLPL LSLLMSSLLALQC +C  DS G C  C  DE+C+WVV TTRD RUP-2  MGKHILLLPLGLSLLMSSLLALQCFRCTSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRD  RUP-4?  GKFVYGNQSCAECNATTVEQGSLIVSTNCCSASHFCNMVYR (ESTs AA945232,AA945121)   GKFVYGNQSCAECNATTVE GSLIVSTNCCSA+ FCNMV+R RUP-2  GKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 101
RUP Homologues Expand a New Sub-family of Secreted Ly-6 Proteins
3D PSSM Fold Recognition Server

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Discriminating Facts from Database Errors in Secreted Ly-6 Proteins

  • 1.
  • 2.
  • 3.
  • 4. Rat Urine  HPLC  Intact MALDI  N-Terminal Sequence High-speed microbore column
  • 5.
  • 6. EST AA893514 vs. dbEST: 30 Rat Hits at 95% to 100% Identity
  • 7.
  • 8. RUP3: Independent MS-based Identification by Wait et al. “Proteins of rat serum, urine and CSF:VI” Electrophoresis 22, 3043-3052 (2001) RUP1 MGKPILLLPLGLSLLMSSLLALQCFR CESLDSTGLCRVGR RICQTYPDEICAWVVVTTRD RUP2 MGKHILLLPLGLSLLMSSLLALQCFR CTSFDSTGFCHVGR QKCQTYPDEICAWVVVTTRD RUP3 MGKHILLLPLGLSLLMSSLLALQCFR CISFDSTGFCYVGR HICQTYPDEICAWVVVTTRD *** *********************** * **** * ***. ****************** RUP1 GKFVYGNQSCAECIGTTVEHGSLIISTNCCSATPFCNMVHP EST AA800439 RUP2 GKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR EST AA893514 RUP3 GKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR EST AA893518 ************* *********.***************
  • 9. RUP Paralogues Define a New Family of Secreted Ly-6 Proteins
  • 10.
  • 11. Three RUP-like Chimeras and a Pre-mRNA L07806 F1-ATPase inhibitor AF368860 UTR F1-ATPase inhib L15618 casein kinase II alpha J03621 mito succinyl-CoA synthase alpha
  • 12. Translation Matches for the Chimeras Reveal a Cryptic Protein RUP-2 28 TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 101 TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 417 TSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 196 L07806 Rattus rattus mitochondrial IF1 protein mRNA RUP-2: 59 RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 101 RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 708 RDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMVHR 580 L15618 Rat casein kinase II alpha subunit (CK2) mRNA RUP-2 24 CFRCTSFDSTGFCHVGRQKCQTYPDEICAWVVVTTRDGKFVYGNQSCAECNATTVEHGSLIVSTNCCSATPFCNMV 99 CF C + +S G C+ C +P E+CA V+T +DGKFVYGNQSCAEC+ TVEHGSLIVSTNCCSAT FCN+V 50 CFECGNLNSMGICNFRTAVCYAHPGEVCA-SVLTYKDGKFVYGNQSCAECSGRTVEHGSLIVSTNCCSATSFCNIV 274 J03621 Rat mitochondrial succinyl-CoA synthetase alpha subunit
  • 14.
  • 15. Multiple Loci on Rat Chromosome 8: Erroneous Mapping of the Chimeras L15618 casein kinase II alpha L07806 F1-ATPase inhibitor AF198441 Rat RUP2 AF198442 Rat spleen protein 1
  • 16.
  • 17.
  • 18. The L07806 Chimera Caused Errors in U niGene
  • 19. RUP Gene Family on Rat 8q21
  • 20. Rat and Mouse RUP Homologues are Highly Diverged
  • 21. Sequences Conserved in Rat but Divergent in Mouse
  • 22. Homologues in Five Mammals but True Orthology Unclear
  • 23.
  • 24. Threading Reveals Homology between RUP1, Lynx1 and Snake Toxin Structures Lynx1, an Endogenous Toxin-like modulator of AChRs in the CNS,
  • 25. Why so Few Apparent Orthologues?
  • 26. P55000 : Antineoplastic Urinary Protein/S ecreted Mammalian Ly-6/uPAR Related Protein – Equivocal Annotation
  • 27.
  • 28. Mouse Ly-6-like Caltrin: Sequence Errors, Unverified Reported Function, New Name and New Function?
  • 29.
  • 30.
  • 31.
  • 32.
  • 33.
  • 34. Human Short Ly6 Proteins None - - 18 + - 11q24.2 113 LVLF31 Genset (sec), USDOH CyC PA2 - 21 + + 11q24.2 126 PATE HGS, ARS, Biovision (partial) Ly6 - 22 + + 8q24.3 103 SLURP1 Genentech, HGS, Incyte Ly6 103 22 + - 8q24.3 125 RGTR43 Genentch (sec/tm) ZymoGenetics Ly6 - 22 + + 8q24.3 97 SLURP2 Curagen, Hyseq, HGS (sec), Incyte (sec) Genset (partial) Ly6 91 19 + + 8q24.3 115 LYNX1 Patents InterPro GPI Sigpep ESTs Ens Chrom Size Name
  • 36. Searches Against Rat ESTs Confirmed the Three mRNAs as Chimeras J03621 L07806 L15618
  • 37.
  • 38. RUP Homologues Expand a New Sub-family of Secreted Ly-6 Proteins
  • 39. 3D PSSM Fold Recognition Server

Notas del editor

  1. AA_DERWENT 1,226,302 sequences;