title: Identification, characterisation and quantification of proteins used in chemical communication
Creators: name: Unsworth, Jennifer
Publisher: name: University of Liverpool Repository
Year: 2015

description: Most animals have excretory systems to remove soluble waste. In humans soluble waste is mainly excreted through the urinary system. Kidneys, urinary bladder and urethra make up this system and are responsible for the production of urine by filtration, reabsorption and secretion. Under normal circumstances urine contains water, creatinine, urea and salts. In humans, the presence of elevated levels of protein or glucose is indicative of medical conditions such as impaired kidney function and diabetes. Some animals are an exception to this. Rodents such as the house mouse (Mus musculus), Norway rat (Rattus norvegicus), bank vole (Myodes glareosin) and Roborovski hamster (Phodopus roborovskii) excrete substantial amounts of protein in their urine yet their renal function remains intact. These proteins belong to the lipocalin family and play an essential part in chemical signalling. Their size (18-19kDa) allows them to escape from being filtered out of the urine during the ultrafiltration step resulting in their excretion in the urine. Many of these proteins share a high sequence identity and genomic data is often incomplete or absent. One aspect of this thesis looks at developing a quantification method for a set of highly homologous lipocalins in mice. Another was to characterise and identify proteins excreted in the harvest mouse (Micromys minutus) and mouse lemur (Microcebus) in the absence of genomic data and see if they are related to the lipocalin family or if they belong to a completely different group of proteins. Using mass spectrometric techniques a method to quantify major urinary proteins (MUPS), lipocalins found in mice, was developed and implemented. A quantification concatemer (QconCAT) was designed to do this and was based on genomic data from the laboratory strain of mouse C57BL/6. MUP isoforms were successfully quantified in both male and female C57BL/6 mice. The QconCAT strategy was also used to assess MUP production during the estrous cycle in female mice. Females express more MUP during the estrous stage with a decline in expression seen during the proestrous. For the second part of this thesis, lipocalin expression in the harvest mouse (Micromys minutus) was investigated. Urine samples collected from male and female harvest mice revealed proteins approximately 18-19 kDa expressed in both sexes. The concentration of protein in urine was much lower than that observed in other rodents. Alternative areas of protein excretion were explored and revealed the same protein to be excreted in much higher concentrations from the saliva and/or paws. Again mass spectrometry was employed to identify and characterise these proteins. A preliminary discovery analysis identified proteins that shared high homology with other lipocalins including MUPS and odorant binding proteins. Intact mass analysis also confirmed the presence of three abundant proteins in both males and females. Anion exchange chromatography was used to separate the proteins for de novo sequence analysis which confirmed that harvest mice excrete proteins belonging to the lipocalin family. The final section of this thesis examines characterising protein expression in the mouse lemur (Microcebus). Although they are classed as primates not rodents, mouse lemurs are known to respond to urinary chemosignals from their conspecifics. Urine samples were collected from two species of mouse lemur - Microcebus murinus and Microcebus lehilahytsara. As mouse lemurs have a specific breeding season samples were collected both in and out of season. Some of the male mouse lemurs from both species expressed a large amount of protein during reproductive season. No protein was observed in females. Intact mass analysis identified a protein at 9388 Da in the M. murinus and 9418 Da in the M. lehilahytsara. Unlike many members of the rodent family who excrete large quantities of lipocalins, de novo sequencing confirmed this protein to be a member of the Whey Acidic Protein family (WAPS). WAPS are expressed across many lineages and have a variety of functions including antibacterial and antifungal action, protease inhibition, tumour suppression and anti-inflammatory activity. No protease inhibition by the mouse lemur protein was observed and further studies will need to be established to determine the biological function of this WAP.