Electrophoretic Analysis of Human Parotid Salivary Proteins with Application to the Study of Rheumatoid Arthritis and Sjogren's Syndrome

Human parotid saliva contains many proteins with diverse functions. In the course of a number of diseases, especially where the normal function of the salivary gland is affected, changes may occur in the levels of certain of these. Therefore the analysis of some of these proteins may be of diagnostic significance. This study has focused on the development and refinement of electrophoretic and of protein detection techniques in order to allow the fractionation of proteins in small volumes of human saliva with the minimum of sample preparation. In order to give an example of their possible diagnostic significance, the electrophoretic techniques which were developed were applied to the fractionation and partial characterisation of the anionic salivary proteins associated with connective tissue disorders such as rheumatoid arthritis and Sjogren's Syndrome. The saliva of patients with rheumatoid arthritis and Sjogren's Syndrome contains additional anionic proteins, which are either present in very low levels or below detection limits in the saliva of normal healthy individuals. Research into the identity of these proteins has been largely hindered by the relatively high electrolyte and low protein content of human parotid saliva, making it necessary to desalt and concentrate the saliva samples prior to carrier ampholyte-based isoelectric focusing. Desalting requires relatively large volumes (preferably > 2ml) of saliva, which may be difficult or even impossible to obtain from diseased glands. Also, one-dimensional isoelectric focusing cannot separate these anionic proteins from the acidic proline-rich proteins of human saliva, as both groups of proteins have overlapping isoelectric points. In this study, a hybrid carrier ampholyte-immobilised pH gradient isoelectric focusing technique was developed to analyse human salivary proteins. Immobilised pH gradients (IPG's) of 3 pH ranges were prepared: broad-range (pH 4-9) IPG was used for the general study of human salivary proteins; while 2 narrow, acidic range IPG's (pH 2.8-4.5 and pH 3.5-5.0) were used to analyse proteins of low isoelectric points, such as the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. This method allowed the difficulties involved when conventional carrier ampholyte-based isoelectric focusing is used to be circumvented, thus making it possible to fractionate the proteins in small volumes (approximately 50ul) of human saliva without prior treatment except for centrifugation. Parotid salivary proteins were also analysed by onedimensional SDS-PAGE. SDS-PAGE gels were subjected to im-muno- and lectin affinity-blotting in order to characterise or identify some of the protein bands. Proline-rich proteins were recognised by their characteristic pink-staining with the dye Coommassie Brilliant Blue R250, and some of the bands which were revealed have been correlated with proline-rich proteins which have been isolated and partially characterised by other research groups. SDS-PAGE failed to reveal any obvious differences between the band patterns of normal subjects and those of patients with rheumatoid arthritis or Sjogren's Syndrome. Two-dimensional gel electrophoresis was also carried out using hybrid carrier ampholyte-immobilised pH gradient polyacrylamide gels in the first dimension and thin-layer SDS-polyacrylamide gradient gels in the second. By means of a combination of staining and electroblotting of the two-dimensional gels onto nitrocellulose followed by probing with specific antisera, a two-dimensional map of human parotid salivary proteins, in which most of the major components have been identified, has been obtained. These techniques were applied to the investigation of the nature of the anionic salivary proteins associated with rheumatoid arthritis and Sjogren's Syndrome. Two-dimensional electrophoresis with pH 3.5-5.0 IPG's in the first dimension followed by silver staining revealed these proteins to be heterogeneous (pI's approximately 3.65-4.75) and of a single relative molecular weight (approximately 32,000). In normal healthy controls these silver stained components were less heterogeneous (pI's approximately 3.65-4.25). Incubation with neuraminidase showed that their heterogeneity was largely due to differing contents of sialic acid in their carbohydrate side-chains. In order to attempt to identify these proteins, the one-dimensional IPG and two-dimensional gels were electroblotted and the blots were probed with a variety of antisera. The proteins appeared to be immunoreactive with antisera to human tissue kallikrein, a protein the level of which has often been reported to be elevated in the saliva of patients with Sjogren's Syndrome. (Abstract shortened by ProQuest.)