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

Abstract

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.

Evolution and Comparative Genomics of Odorant- and Pheromone-Associated Genes in Rodents

“…Two Ly-6 homologous proteins were originally identified in rat urine (Southan et al 2002). Mapping their genes to the same region of the rat genome resulted in the identification of a large family of Ly-6 homologous proteins, which we shall refer to as Ly-6-homologous urinary proteins (LUPs).…”

“…Like MUPs they possess the unusual property of being present at high levels in male rat urine (Southan et al 2002). Moreover, their rapid sequence diversification, pseudogenization and gene duplication in rodents mirror those seen for MUPs and are strong evidence for adaptive evolution.…”

SSLP-1, a secreted Ly-6 protein purified from mouse seminal vesicle fluid

“…The results of Edman sequencing suggest that Asn 10 is one N-glycosylated site. In contrast with these findings for SSLP-1, SVS7 (Luo et al 2001), and SLURP-1 (Chimienti et al 2003) are not glycoproteins, while RUP-1 and RUP-2 have only one predicted N-glycosylation site, and the evidence suggesting that they are glycosylated is indirect (Southan et al 2002). The function of glycosylation in these secreted proteins remains to be determined.…”

“…GPI-anchored Ly-6 receptor proteins are thought to be involved in cellular adhesion and signaling (Gumley et al 1995, Bamezai 2004). The second non-GPI-anchored subfamily contains secreted Ly-6 proteins, including secreted mammalian Ly-6/uPARrelated protein (SLURP)-1 (Adermann et al 1999), SLURP-2 (Tsuji et al 2003), rat urinary protein (RUP)-1, RUP-2, pig protein (PIP)-1, and rat spleen protein (RSP)-1 (Southan et al 2002), and seminal vesicle secretion 7 (SVS 7; Southan et al 2002). SLURP-1 in human blood and urine was the first of the secreted Ly-6 protein to be discovered (Adermann et al 1999).…”

“…While it is known that they are secreted and glycosylated proteins, their biological functions are obscure (Southan et al 2002). SVS7 was originally purified from the mouse seminal vesicle and has caltrin activity inhibiting calcium uptake by epididymal sperm (Coronel et al 1992).…”