Genomic/proteomic analysis of human salivary glands

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The overall goal of Subproject 3 is to compare the expression of proteins and transcripts in normal human parotid gland with parotid tissue derived from individuals with idiopathic dry mouth. The overall hypothesis is that idiopathic dry mouth is caused by a change in the expression of key regulatory or effector proteins. To identify human parotid gland-specific proteins, in Specific Aim 1: we will create an expressed-sequence-tag [EST] data base of the human parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland. To accomplish this goal, we will construct a directional cDNA library from normal human adult parotid gland tissue and will sequence the 5' end of randomly selected clones. Sequences will then be compared against available databases. We hypothesize that idiopathic dry mouth results from a change in the expression of key regulatory or effector proteins leading to dysregulation of fluid production. To test this hypothesis, in Specific Aim 2: we will compare the expression of transcripts in normal glands with those derived from individuals with idiopathic dry mouth using microarrays and direct sequencing of PCR amplified transcripts. As a complement to the EST database outlined in Specific Aim 1, we propose in Specific Aim 3, to map the spectrum of proteins which are secreted by the normal human parotid and submandibular/sublingual glands. Two dimensional gel electrophoresis will be used to separate proteins and combinations of immunoblotting, Edman degradation and matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) mass spectrometry will be used to identify the proteins. We hypothesize that differences will be observed between this "normal" profile and one obtained using saliva collected from subjects with idiopathic dry mouth, who present with seemingly "normal" flow rates. Proteins displaying variance in their level for expression will be candidates for functional roles involved in maintaining the oral cavity in a lubricated and hydrated state. Taken together, these approaches will allow us to identify underlying defects in key regulatory or effector proteins which lead to oral dryness.