Secretory cargo composition affects polarized secretion in MDCK epithelial cells.

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a "sorting escort" (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as "sorting escorts" to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells.

N-terminal proteolytic processing of porcine chromogranin A in parathyroid tissue.

Chromogranin A (CgA) is a glycoprotein stored in secretory granules of many endocrine and neuroendocrine cells. CgA undergoes tissue specific processing to release regulatory peptides. In the parathyroid, although processing is limited and variable, several CgA-derived peptides have been characterized including parastatin and betagranin. An early stage of CgA processing is the generation of a 64-kDa fragment (CgA64). In this study, we have purified CgA64 from porcine parathyroid glands by chromatographic separations. Edman degradation of this CgA64 yielded the N-terminal sequence NDQAELKEGTEEASSKEAAEKRGDXAVEKND corresponding to pCgA(94-125). Amino acid composition suggests that CgA64 corresponds to CgA(94-430) (i.e. the entire CgA molecule, less the N-terminal residues 1-93). To determine the origin of CgA64, we fractionated parathyroid membrane vesicles by sucrose gradient centrifugation. Intact CgA is predominantly located in dense sucrose fractions (secretory granules), whereas CgA64 is located near the top of the gradient (soluble protein fraction). In vitro incubation of these fractions revealed that the conversion of CgA did not occur in intact granules. These results indicate that CgA64 is not present in intact granules suggesting that it is not a naturally occurring secretory product in parathyroid cells.