PH- and Ca(2+)-induced Conformational Change and Aggregation of Chromogranin B. Comparison with Chromogranin A and Implication in Secretory Vesicle Biogenesis

Chromogranins A and B have been known to undergo pH- and Ca(2+)-dependent aggregation, and this property is considered essential for the proper sorting of the vesicular matrix proteins. In the present study, purified native chromogranin B (CGB) from bovine adrenal medulla was used to study the pH- and Ca(2+)-dependent conformational changes and aggregation property. Similar to chromogranin A (CGA), which had been shown to undergo pH- and Ca(2+)-dependent conformational changes and to be composed of 60-65% random coil with 25-40% alpha-helicity, chromogranin B was also shown to consist of 65-70% random coil, 15-25% alpha-helix, and 10-15% beta-sheet structures. The high percentage of random coil suggests that CGB behaves hydrodynamically as an asymmetric molecule, thus explaining its anomalous migration on SDS-polyacrylamide gels. Further, CGB eluted from a gel filtration column in the volume indicative of a globular protein with molecular weight of approximately 200,000 at both the intravesicular pH of 5.5 and a near physiological pH of 7.5. Considering that dimeric CGA eluted from a gel filtration column in the position suggestive of a 300-kDa protein, this result indicated that CGB exists in a monomeric state at both pH levels. Like CGA, which exhibited greater aggregation at pH 5.5 than at pH 7.5 upon Ca2+ binding, CGB also aggregated much more readily at pH 5.5 than at pH 7.5. However, there was a marked difference in the aggregation properties of CGA and CGB with regard to their sensitivity to Ca2+: CGB was at least 2 orders of magnitude more sensitive to Ca2+ than CGA. This suggested that, in spite of the low concentration of CGB (approximately one-tenth that of CGA) in bovine adrenal chromaffin cells, CGB would start to aggregate well ahead of CGA in the trans-Golgi network. In view of the proposed importance of the pH- and Ca(2+)-induced chromogranin aggregation in vesicle biogenesis, the extreme sensitivity of CGB aggregation to Ca2+ appears to underline the potential importance of CGB aggregation in the early stages of vesicle biogenesis.