The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data.
A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.
Southan
Christopher
C
Department of Bioinformatics, Glaxo SmithKline Pharmaceuticals, Harlow, UK. chris.southan@ogs.co.uk
Cutler
Paul
P
Birrell
Helen
H
Connell
John
J
Fantom
Kenneth G M
KG
Sims
Matthew
M
Shaikh
Narjis
N
Schneider
Klaus
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D020224:Expressed Sequence Tags; D006031:Glycosylation; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D020543:Proteome / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014556:Urine / Q000737:chemistry
D020543:Proteome; D012333:RNA, Messenger