Proteomic Analysis of Zymogen Granules

María Gómez-Lázaro; Cornelia Rinn; Miguel Aroso; Francisco Amado; Michael Schrader

Disclosures

Expert Rev Proteomics. 2010;7(5):735-747. 

In This Article

Proteomics of Related Regulated Secretory Proteins: Salivary Granules & Saliva

The development of proteomics technologies has allowed the characterization of other regulated secretory organelles and proteins such as neutrophil granules and synaptic vesicles (for a comprehensive review, see [104]) as well as the analysis of salivary secretions. Whole saliva is a complex mixture that comprises the secretion of salivary glands in addition to the contribution of bacteria, serum and cellular debris.[105,106] There is currently great interest in salivary biomarker research and technology developments to exploit saliva for diagnosis and other clinical applications.[107,108] Whole saliva presents a large array of proteins and peptides participating in a multitude of functional roles in the oral cavity, such as microbiological protection, lubrication, digestion and general maintenance of teeth and mucosal surface health.[105,109] Traditional proteomic methods were applied to the analysis of whole-saliva peptides/protein species using 2D-SDS-PAGE followed by MS. With this approach, the most abundant salivary proteins were identified, such as several proline-rich proteins (PRPs), amylase, lipocalin-1 or calgranulin B, assigning more than 300 proteins (reviewed in [105,106,110]). The presence of high amounts of mucins and other glycoproteins interferes with 2D protein separation by blocking the passage of current during the focusing step, and by forming heterotypic complexes with other salivary proteins.[111] The use of multidimensional liquid chromatographic systems coupled to MS allowed a dramatic increase to more than 1000 salivary proteins being identified.[112]

The components of saliva are mainly produced by acinar cells and are conveyed to the oral cavity by a cell-lined duct system in which the fluid and electrolyte components are subject to secondary modifications. Secreted salivary proteins are predominantly stored at high concentrations in dense-core secretory granules (reviewed in[16,113,114]). Despite a large number of studies devoted to the characterization of the dense-core granules, information about their composition remains limited. The general procedure for secretory granule isolation is based on homogenizing the gland tissue (final 5% w/t) in buffered sucrose solution (in the range of pH 6.5–7.6, required to maintain intact granules) and supplemented with EDTA (preventing organelle aggregation). Following this, a secretory granule fraction with high purity is obtained by submitting the homogenate supernatant to a sucrose/ficoll gradient,[115] sucrose gradient[116,117] or Percoll gradient.[118,119] In the case of parotid acinar cells, isolation of secretory granules can be performed by differential centrifugation,[120] since secretory granules are dense and of large size. Moreover, density-gradient separation allowed the isolation of well-defined secretory granule populations indicative of differences in granule content, protein distribution or degree of maturation.[119,121]

Interestingly, a few proteins known to be present in parotid secretory granules have been identified in ZGs, as well as in recent proteomic studies.[34,35] These include Rac26, Noc2, Rab27B, Rap1 and Rab3D. Enlarged granules with the granule volume being doubled have been observed in both the exocrine pancreas and parotid gland in Rab3D-knockout mice, pointing to some overlap in the machinery responsible for regulated exocrine secretion.[122]

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