©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
pH- and Ca-induced Conformational Change and Aggregation of Chromogranin B
COMPARISON WITH CHROMOGRANIN A AND IMPLICATION IN SECRETORY VESICLE BIOGENESIS (*)

Seung Hyun Yoo (§)

From the (1) Laboratory of Neurochemistry, NIDCD, National Institutes of Health, Bethesda, MD 20892-3320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chromogranins A and B have been known to undergo pH- and Ca-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-dependent conformational changes and aggregation property. Similar to chromogranin A (CGA), which had been shown to undergo pH- and Ca-dependent conformational changes and to be composed of 60-65% random coil with 25-40% -helicity, chromogranin B was also shown to consist of 65-70% random coil, 15-25% -helix, and 10-15% -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 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 Ca 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 Ca: CGB was at least 2 orders of magnitude more sensitive to Ca 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-induced chromogranin aggregation in vesicle biogenesis, the extreme sensitivity of CGB aggregation to Ca appears to underline the potential importance of CGB aggregation in the early stages of vesicle biogenesis.


INTRODUCTION

Chromogranins A and B are found in the secretory vesicles of virtually all neuronal and endocrine cells (Simon and Aunis, 1989; Winkler and Fischer-Colbrie, 1992). They are co-stored with hormones, ions, neurotransmitters, and other proteins and peptides in the secretory vesicles (Winkler et al., 1986). Both chromogranins are acidic proteins with isoelectric points of 4.5-5.5 (Fischer-Colbrie and Schober, 1987; Simon and Aunis, 1989; Winkler and Fischer-Colbrie, 1992), and both bind Ca (Bulenda and Gratzl, 1985; Reiffen and Gratzl, 1986). Chromogranin A is a high capacity, low affinity Ca-binding protein, binding up to 32-55 mol of Ca/mol of protein with dissociation constants of 2-4 mM (Yoo and Albanesi, 1991), and is suggested to be responsible for the inositol 1,4,5-trisphosphate-sensitive intracellular Ca store role of the secretory vesicles of adrenal chromaffin cells (Yoo and Albanesi, 1990a). Furthermore, chromogranins A and B were shown to interact with the secretory vesicle membrane at the intravesicular pH of 5.5 and to be released from it at a near physiological pH of 7.5 (Yoo, 1993a, 1993b). In view of the fact that chromogranin A was shown to exist in a dimeric state at pH 7.5 and in a tetrameric state at pH 5.5 (Yoo and Lewis, 1992), it appears that tetrameric CGA() interacts with the protein component(s) of the vesicle membrane at the intravesicular pH of 5.5 (Yoo, 1993a). Subsequent studies indicated that CGA interacts with the integral membrane proteins of the vesicle membrane, and one of the CGA-interacting membrane proteins was tentatively identified to be the inositol 1,4,5-trisphosphate receptor/Ca channel (Yoo, 1994).

In addition, chromogranins A and B are known to aggregate at acidic pH levels in the presence of Ca (Gerdes et al., 1989; Gorr et al., 1989; Yoo and Albanesi, 1990b; Chanat and Huttner, 1991), and this pH- and Ca-dependent aggregation property of chromogranins has been postulated to be essential for a proper sorting of vesicle matrix proteins (Gerdes et al., 1989; Gorr et al., 1989; Yoo and Albanesi, 1990b; Chanat and Huttner, 1991). The aggregation of CGA was shown to be highly dependent on both pH and Ca concentration; at a given Ca concentration, the extent of CGA aggregation was very high at pH 5.5, whereas there was considerably less aggregation at pH 7.5 (Yoo and Albanesi, 1990b). The rate and extent of aggregation were directly dependent on Ca concentration at pH 5.5, demonstrated by the fact that the rate of aggregation was faster and the extent greater at higher Ca concentrations (Yoo and Albanesi, 1990b). The aggregation of CGA at pH 5.5 appeared to be so nearly instantaneous that the aggregation of CGA was virtually complete in the first few seconds after the introduction of Ca (Yoo and Albanesi, 1990b). Moreover, circular dichroism data indicated that CGA undergoes pH- and Ca-dependent conformational changes (Yoo and Albanesi, 1990b) and that the protein is comprised largely of random coil (60-65%) with some -helix (25-40%) (Yoo and Albanesi, 1990b).

Although purified CGB has previously been obtained either by elution from SDS-polyacrylamide gels (Fischer-Colbrie and Schober, 1987; Benedum et al., 1987; Gorr et al., 1989; Gill et al., 1991) or by employing a boiling step (Fischer-Colbrie and Schober, 1987), which is known to change the native conformation of chromogranin A (Yoo and Albanesi, 1990b), chromogranin B has not been purified in its native state so far. As part of efforts to elucidate the biochemical property of CGB, purified native CGB was studied using fluorescence and circular dichroism spectroscopy, gel filtration chromatography, and pH- and Ca-dependent aggregation study. Unlike chromogranin A, which was found to exist in a dimeric or tetrameric state depending on pH (Yoo and Lewis, 1992), chromogranin B appears to exist entirely in a monomeric state. Moreover, chromogranin B exhibited a pH- and Ca-dependent aggregation property quite different from that of chromogranin A (i.e. CGB started to aggregate at protein or Ca concentrations that were at least 2 orders of magnitude lower than those shown with CGA). Although the aggregation property of CGB is in line with the proposed roles of chromogranins A and B in vesicle biogenesis (Huttner et al., 1988 and 1991; Yoo, 1993a and 1993b), the difference in the aggregation property appears to underline the subtle but crucial differences in the roles CGA and CGB might play during vesicle biogenesis.


EXPERIMENTAL PROCEDURES

Materials

4,4`-Bis(1-anilinonaphthalene 8-sulfonate) (bis-ANS) was from Molecular Probe. Sephacryl S-300 and molecular size markers (except calmodulin) were obtained from Pharmacia Biotech Inc. Chelex 100 and acrylamide were from Bio-Rad. Sodium acetate, EGTA, and MOPS were from Sigma.

Chromogranin and Calmodulin Preparation

Chromogranin A was purified using the vesicle lysates of bovine adrenal medullary chromaffin cells as the starting material as described previously (Yoo and Albanesi, 1990b), and chromogranin B was also purified from the vesicle lysates utilizing several chromatographic and electrophoretic steps.() Calmodulin from bovine brain was purified according to the method of Dedman and Kaetzel(1983).

Fluorescence Spectroscopy

Steady-state fluorescence measurements were carried out using a DMX-1000 spectrofluorometer (SLM-AMINCO). Excitation and emission bandpasses were set at 8 nm. All fluorescence measurements were made at 22 °C using a dual path length ``T-cuvette'' with the short path length (2 mm) oriented toward the excitation side to minimize inner filter effects.

CD Spectroscopy

CD spectra were recorded using a Jasco (Easton, MD) 600 circular dichroism spectrophotometer using a cell with a path length of 0.2 mm and a protein concentration of 0.08 mg/ml in either 7.5 mM sodium acetate, pH 5.5, or 7.5 mM MOPS, pH 7.5. All spectra were taken at 22 °C and are the averages of at least four scans. Analysis of the spectra was carried out using the polylysine spectra of Greenfield and Fasman(1969) as a standard.

Aggregation Experiments

Chelex 100-treated chromogranin B or chromogranin A in either 15 mM sodium acetate, pH 5.5, or 15 mM MOPS, pH 7.5, was titrated with concentrated CaCl. Aggregation was monitored by measuring the turbidity change at 320 nm with a Beckman DU 70 spectrophotometer. All the measurements were done at 22 °C.

Other Methods

Concentration of bis-ANS in ethanol was determined spectrophotometrically at 385 nm using 16,670 mol cm as the extinction coefficient (Farris et al., 1978). Protein determination was according to Bradford(1976).


RESULTS

The purified native CGB was obtained by subjecting the secretory vesicle lysates of bovine adrenal medullary chromaffin cells to several chromatographic and electrophoretic steps (Fig. 1) and was used in the present experiments. Although chromogranins are highly charged acidic proteins, CGA was shown to possess hydrophobic regions, as demonstrated by the increase in fluorescence emission upon binding the fluorescence probe, bis-ANS (Rosen and Weber, 1969). bis-ANS is known to bind to the hydrophobic regions of proteins and to increase its fluorescence emission markedly upon binding to the hydrophobic regions (Rosen and Weber, 1969). In order to determine whether CGB also has hydrophobic regions, the changes in bis-ANS fluorescence were measured upon introduction of purified native CGB. As shown in Fig. 2, the fluorescence emission of bis-ANS shows a marked enhancement and blue shift upon introduction of CGB. The emission due to 2 µM bis-ANS, shown in spectrum A, exhibits a maximum near 498 nm. However, the emission maximum shifts to 480 nm (spectrum B) upon introduction of 1 µM CGB, and the yield (calculated from the areas under the spectra) increased approximately 50-fold (the ordinates for spectra A and B are accordingly different). The inset shows the titration of 1 µM CGB with bis-ANS, demonstrating a marked enhancement of fluorescence intensity and a saturation effect. Unlike CGA, which had shown the increase in fluorescence signal upon the introduction of Ca at the saturating level of bis-ANS (40 µM) (Yoo and Albanesi, 1990b), it was not readily apparent what effect Ca has on the fluorescence change of bis-ANS-CGB due to the extreme sensitivity of the Ca-induced aggregation of CGB to Ca (see below).


Figure 1: SDS-polyacrylamide gel electrophoresis of the vesicle lysates and purified chromogranin B. The vesicle lysate proteins and purified CGB from bovine adrenal medullary chromaffin cells were visualized on a 10% SDS-polyacrylamide gel. LaneA, vesicle lysate protein (14 µg); laneB, purified intact CGB (0.6 µg). Chromogranins A and B are indicated by arrows.




Figure 2: Fluorescence emission spectra showing the increase in quantum yield upon binding of bis-ANS to chromogranin B. Fluorescence emission spectra of either 2 µM bis-ANS only (A) or 2 µM bis-ANS plus 1 µM chromogranin B (B) are shown. SpectrumA was drawn on an expanded scale. Excitation was at 400 nm, and the buffer used was 20 mM sodium acetate, pH 5.5. Inset, titration of 1 µM chromogranin B with bis-ANS until the fluorescence signal plateaued. Excitation, 420 nm; emission, 480 nm.



To determine the secondary structure of CGB, CGB was studied by CD spectroscopy. The CGB spectra in Fig. 3show that CGB exhibits different CD spectra at pH 5.5 and 7.5, indicating that CGB undergoes a pH-dependent conformational change. In the absence of Ca, the change of pH from 7.5 to 5.5 increased the -helicity from 15 to 20% based on the Greenfield and Fasman approximation(1969), although the random coil content remained the same at 70% (). As had been observed with CGA, Ca has also changed the CD spectra of CGB at pH 5.5. Addition of 0.2 mM Ca at pH 5.5 further increased the -helicity from 20 to 25%, whereas the random coil decreased from 70 to 65%. Because the estimated -helicity of 15-25% for CGB is lower than that of CGA (25-40%) and the random coil content of 65-70% for CGB is higher than that of CGA (60-65%) (Yoo and Albanesi, 1990b), CGA was subjected to CD spectroscopy in the presence of 0.2 mM Ca at pH 5.5 in order to obtain the secondary structure information under identical conditions. The results in Fig. 4 show a marked difference in the spectra of CGB and CGA under identical conditions. Analysis of the CGA spectra by the same method (Greenfield and Fasman, 1969) shows that CGA has 40% -helicity, 0% -sheet, and 60% random coil in the presence of 0.2 mM Ca at pH 5.5 (), agreeing with the previous results obtained in the absence of Ca at pH 5.5 (Yoo and Albanesi, 1990b). The absence of the effect of submillimolar Ca on CGA secondary structure has already been observed in our previous study in which Ca was able to induce the conformational changes of CGA only at millimolar Ca concentrations (10 mM or higher) (Yoo and Albanesi, 1990b).


Figure 3: CD spectra of chromogranin B at pH 5.5 and 7.5. A, chromogranin B in 7.5 mM MOPS, pH 7.5, and 0.2 mM EGTA; B, CGB in 7.5 mM sodium acetate, pH 5.5, and 0.2 mM EGTA; C, CGB in 7.5 sodium acetate, pH 5.5, and 0.2 mM CaCl. Chromogranin B concentration was 0.08 mg/ml.




Figure 4: CD spectra of chromogranin A and chromogranin B at pH 5.5. Chromogranin B (solid line) and chromogranin A (dotted line) in 7.5 mM sodium acetate, pH 5.5, and 0.2 mM CaCl are shown. Chromogranin B and chromogranin A concentrations were 0.08 mg/ml (1.1 µM) and 0.11 mg/ml (2.3 µM), respectively.



In light of the fact that CGA exists in a dimeric or tetrameric state (Yoo and Lewis, 1992) and dimeric CGA elutes from a gel filtration column in the elution volume indicative of a globular protein with a molecular weight of 300,000 (Yoo and Albanesi, 1990b, 1991), chromogranin B was also subjected to gel filtration chromatography to estimate the oligomerization state of the molecule. As shown in Fig. 5, CGB eluted in the volume characteristic of a globular protein with molecular weight of either 220,000 at pH 7.5 or 170,000 at pH 5.5. These estimated values are far below that found for dimeric CGA (300,000). Therefore, considering the fact that monomeric CGA and CGB migrate as proteins with masses of 75 and 110 kDa, respectively, on SDS-polyacrylamide gel electrophoresis (Fig. 1), the estimated CGB mass of 200 kDa by gel filtration chromatography (Fig. 5) may simply reflect the highly random, markedly asymmetric structure of CGB and strongly suggests that CGB exists in a monomeric state at both pH 5.5 and 7.5.


Figure 5: Gel filtration on Sephacryl S-300. Chelex 100-treated chromogranin B was chromatographed on a calibrated Sephacryl S-300 column (0.9 47 cm) equilibrated with either 15 mM Tris-HCl, pH 7.5, or 15 mM sodium acetate, pH 5.5, both in 0.1 M KCl, 1 mM dithiothreitol, and 2 mM EGTA and eluted at 7.2 ml/h. The column was calibrated with molecular markers, blue dextran for void volume (V, 2 10 Da), ferritin (440 kDa), aldolase (158 kDa), and calmodulin (17 kDa). The elution locations of chromogranin B at pH 5.5 (A) and at pH 7.5 (B) are indicated by arrows.



In view of the pH- and Ca-dependent aggregation of CGA, the pH- and Ca-dependent aggregation property of CGB has also been studied by measuring Ca-induced turbidity changes. As shown in Fig. 6, Ca induced aggregation of CGB at various protein concentrations, and the aggregation pattern was dependent upon the protein concentration; a lower concentration of Ca was required to induce aggregation as CGB concentration was increased. The extent of aggregation was also strongly dependent on Ca concentration at each CGB concentration. When the chromogranin B concentration was low, the maximal degree of aggregation was also low, thus indicating a critical dependence of the aggregation on the CGB concentrations. Moreover, the rate of aggregation at a given CGB concentration was directly dependent on Ca concentration (Fig. 7).


Figure 6: Aggregation of chromogranin B as a function of Ca concentration. Chromogranin B concentrations are 0.4 (), 0.15 (), 0.06 (), and 0.02 mg/ml (). The A values shown are those taken 2 min after the addition of Ca. The dashedportion of each curve indicates that the aggregation peaked and the aggregated protein started to precipitate out of the solution.




Figure 7: Time course of chromogranin B aggregation. The aggregation was monitored after adding 10 (), 3 (), or 1 mM CaCl () to 0.06 mg/ml CGB in 20 mM sodium acetate, pH 5.5.



Because CGA aggregation was highly dependent on pH (Yoo and Albanesi, 1990b), the effect of pH on CGB aggregation was also studied. As shown in Fig. 8, the extent of aggregation was much higher at pH 5.5 than at pH 7.5; even at a maximal level of aggregation, the extent of aggregation at pH 7.5 was less than half that at pH 5.5, clearly demonstrating the favorable aggregation of CGB at the intravesicular pH of 5.5. Analogous to the results obtained with CGA (Yoo and Albanesi, 1990b), chromogranin B also exhibited a markedly different Ca sensitivity at the two pH levels; at pH 5.5 the aggregation was completed at 8 mM Ca, but, at the same Ca concentration (8 mM), the aggregation at pH 7.5 was only half of the maximal aggregation, which occurred at 16-17 mM Ca. The extent and Ca dependence of CGB aggregation at pH 5.5 appeared to be significantly different from those of CGA (Yoo and Albanesi, 1990b). Hence, in order to directly compare the aggregation property of CGA and CGB, the aggregation of CGA as a function of Ca was measured. As shown in Fig. 9, 0.4 mg/ml CGB started to aggregate upon introduction of submillimolar Ca at pH 5.5, and the aggregation was completed at 3-4 mM Ca, whereas the aggregation of the same amount of CGA virtually did not even start at 10 mM Ca. However, CGA aggregation became appreciable at 20 mM Ca and peaked at 38-40 mM Ca, agreeing with the previous results, in which 0.6 mg/ml CGA aggregated maximally at approximately 38 mM Ca (see Fig. 5of Yoo and Albanesi, 1990b). Even at its maximal aggregation, the extent of CGA aggregation was less than one-sixth of that observed with the same amount of CGB.


Figure 8: Effect of pH on chromogranin B aggregation. Aggregation of CGB (0.15 mg/ml) at pH 5.5 () and 7.5 () was measured as a function of Ca concentration. The buffers were either 20 mM sodium acetate, pH 5.5, or 20 mM MOPS, pH 7.5. The A values are those taken 2 min after the addition of Ca.




Figure 9: Comparison of chromogranin A and chromogranin B aggregation as a function of Ca concentration. Protein concentration was 0.4 mg/ml in 15 mM sodium acetate, pH 5.5. The A values for CGA () and CGB () are those taken 2 min after the addition of Ca.



Further, in order to determine whether the presence of both CGA and CGB affects the aggregation property of each chromogranin, the Ca-induced aggregation of the mixture of CGA and CGB was studied. As shown in Fig. 10, the CGA/CGB mixture aggregated maximally at a Ca concentration that lies between the two Ca concentrations needed for the maximal aggregation of CGA and CGB, respectively; when 0.06 mg of CGB/ml was mixed with 0.4 mg of CGA/ml, a CGB/CGA ratio similar to that found in secretory vesicles, the mixture exhibited a maximal aggregation at 30 mM Ca, which is higher than the 12 mM Ca needed for a maximal aggregation of 0.06 mg of CGB/ml but lower than the 40-45 mM Ca needed for a maximal aggregation of 0.4 mg of CGA/ml. In another experiment, when a larger amount of CGB (0.15 mg of CGB/ml) was mixed with the same amount of CGA (0.4 mg/ml), the aggregation of the CGB/CGA mixture showed a maximal aggregation at 13 mM Ca, which is higher than the 8 mM Ca needed for the maximal aggregation of 0.15 mg of CGB/ml (Fig. 6) but significantly lower than the 30 mM Ca needed for the maximal aggregation of the mixture of 0.06 mg of CGB/ml and 0.4 mg of CGA/ml (Fig. 10), suggesting that the Ca sensitivity of the aggregation of the CGB/CGA mixture is related to the relative ratio of CGB over CGA in the mixture.


Figure 10: Aggregation of chromogranin A and chromogranin B mixture as a function of Ca concentration. Aggregation of the mixture of CGB (0.06 mg/ml) and CGA (0.4 mg/ml) in 15 mM sodium acetate, pH 5.5, was measured as a function of Ca concentration. The A values for CGB alone (), the CGA-CGB mixture (), and CGA alone () are those taken 2 min after the addition of Ca.




DISCUSSION

As had been observed with chromogranin A (Yoo and Albanesi, 1990b), the present results demonstrate that chromogranin B has hydrophobic regions and also undergoes pH- and Ca-dependent conformational changes. However, unlike CGA, which had been shown to exist in a dimeric state at a near physiological pH of 7.5 and in a tetrameric state at the intravesicular pH of 5.5 (Yoo and Lewis, 1992), CGB appeared to exist only in a monomeric state at both pH 5.5 and 7.5 (Fig. 5).

Analogous to chromogranin A, which had been shown to contain 60-65% random coil with 25-40% -helix and 0-10% -sheet structures (Yoo and Albanesi, 1990b), CGB was also shown to consist largely of random coil (65-70%) with 15-25% -helix and 10-15% -sheet structures. The change of pH from 7.5 to 5.5 in the absence of Ca increased the -helicity from 15 to 20%, whereas the random coil content remained the same at 70%. The addition of 0.2 mM Ca to CGB at pH 5.5 further increased the -helicity from 20 to 25% while decreasing the random coil content from 70 to 65%. Although the introduction of Ca to CGA at pH 5.5 has also changed the conformation of CGA (Yoo and Albanesi, 1990b), there was no detectable conformational change of CGA in the low range of Ca concentration; the conformational change of CGA was only seen at Ca concentrations of 10 mM or higher (Yoo and Albanesi, 1990b). However, in the case of CGB, submillimolar Ca was sufficient to cause conformational changes of CGB ( Fig. 3and ). In fact, higher concentrations of Ca caused aggregation of CGB (Fig. 6-8). Considering that CGB binds to the vesicle membrane at pH 5.5 (Yoo, 1993b), the increase of -helicity at pH 5.5 might contribute to the pH-dependent membrane binding of CGB. Further, the presence of a high percentage of random coil (65-70%) suggests that CGB is probably a markedly asymmetric molecule, contributing to its anomalous migration on SDS-polyacrylamide gel electrophoresis in that it migrates significantly more slowly than globular molecules of its size. In our previous study, dimeric CGA eluted on gel filtration chromatography in the volume suggestive of a 300-kDa protein (Yoo and Albanesi, 1990b, 1991), which is significantly larger than its actual mass of 96 kDa (2 48 kDa). Therefore, in view of the fact that the molar mass of bovine CGA is 48 kDa (Iacangelo et al., 1986; Benedum et al., 1986) and that of bovine CGB is 71 kDa (Bauer and Fischer-Colbrie, 1991), it is reasonable to assume that CGB exists in a monomeric state based on its elution position on gel filtration chromatography, where it eluted in the volume indicative of a globular protein with a molecular weight of 200,000.

The aggregation of CGB was highly dependent on pH and Ca as was the case with CGA. However, there were large differences in the extent of aggregation and the sensitivity to Ca concentrations between CGA and CGB. When an equal amount of protein was used, 10-fold higher Ca was needed to induce a maximal aggregation of CGA than was needed for a maximal aggregation of CGB (Fig. 9) (i.e. approximately 40 mM Ca was needed to induce a maximal aggregation of 0.4 mg/ml CGA (A reading of 0.27), whereas only approximately 4 mM Ca was needed to induce a maximal aggregation of the same amount of CGB (A reading of 1.72)). Nevertheless, the extent of maximal CGA aggregation was only one-sixth that of CGB under identical conditions (Fig. 9). The difference in Ca sensitivity of aggregation between CGA and CGB can be demonstrated more clearly by comparing the concentrations of Ca needed to induce the same degree of aggregation with an equal amount of each protein. For example, to induce the CGB aggregation with an A reading of 0.2, 0.4 mg/ml CGB required 0.2 mM Ca at the intravesicular pH of 5.5, whereas an equal amount of CGA required 33 mM Ca to induce the same level of aggregation (Fig. 9). This result suggests that the aggregation of CGB is approximately 165-fold more sensitive to Ca than CGA. The difference in Ca sensitivity between CGA and CGB aggregation might be due to the fact that 1) a lower Ca concentration can cause larger conformational changes in CGB than possible in CGA (Tables I and II) (Yoo and Albanesi, 1990b), thereby possibly exposing more hydrophobic regions in CGB, which may participate in the aggregation of CGB, or 2) CGA exists in a tetrameric state at pH 5.5 (Yoo and Lewis, 1992) compared with the monomeric existence of CGB (Fig. 5), which will severely restrict the orientation of CGA molecules toward each other, thus reducing the possibility of the potential interacting regions from interacting with each other.

Furthermore, by comparing the amount of protein needed to induce a given level of protein aggregation at a fixed Ca concentration, the degree of sensitivity of each CGA and CGB aggregation can also be determined. As shown in Fig. 6 , 0.02 mg/ml CGB required 12 mM Ca to induce its aggregation to the extent that A = 0.1, whereas 3.2 mg/ml CGA was needed at the same Ca concentration (12 mM) to induce the same level of aggregation (see Fig. 5of Yoo and Albanesi (1990b)). In other words, 160-fold more CGA was required at a given Ca concentration to induce the same degree of aggregation that was achieved with CGB. In light of the observation that a higher degree of chromogranin aggregation is achieved in the presence of a larger amount of chromogranin at a given Ca concentration (Fig. 6) (Yoo and Albanesi, 1990b), the above results clearly demonstrate the fact that CGB aggregation at the intravesicular pH of 5.5 is at least 2 orders of magnitude more sensitive than that of CGA at a given Ca concentration. This result in turn suggests that CGB would start to aggregate in the low pH and high Ca environment of the intravesicular milieu even at a concentration less than one-hundredth that of CGA, although aggregation of CGB will be affected by the presence of CGA and other proteins (Fig. 10). In particular, in view of the observation that CGA and CGB bind to each other at the intravesicular pH of 5.5 (Yoo, 1995), the intermediate Ca sensitivity of the aggregation of the CGA/CGB mixture (Fig. 10) appears to be an inevitable result of two interacting proteins, each with a different Ca sensitivity. Nevertheless, in spite of the fact that the amount of CGB in the secretory vesicles of bovine adrenal chromaffin cells is approximately one-tenth that of CGA (Fischer-Colbrie and Schober, 1987; Winkler and Fischer-Colbrie, 1992), the exceedingly Ca-sensitive nature of CGB aggregation would more than compensate for the low CGB concentration, resulting in the aggregation of CGB ahead of CGA in the trans-Golgi network.

Given that pH- and Ca-induced aggregation of chromogranins A and B has been proposed to play vital roles in sorting the vesicle matrix proteins in the trans-Golgi network (Gerdes et al., 1989; Gorr et al., 1989; Yoo and Albanesi, 1990b; Chanat and Huttner, 1991), the present results provide strong evidence in support of such proposals. In particular, given that the sorting of the potential vesicular matrix proteins is considered an essential step during vesicle biogenesis, the aggregation of CGB, probably well ahead of CGA in the trans-Golgi network where secretory vesicles are formed, may prove to be a critical first step in the sorting of the vesicular matrix proteins. Recently, chromogranin B was also shown to bind to the vesicle membrane at the intravesicular pH of 5.5 and to be released from it at a near physiological pH of 7.5 (Yoo, 1993b). Further, chromogranin B was shown to interact with the vesicle membrane significantly more tightly than CGA. Therefore, in light of the present results, which suggest the aggregation of CGB prior to that of CGA in the trans-Golgi network, it is likely that the CGB aggregate would begin to interact with the vesicle membrane well ahead of CGA during vesicle biogenesis, thereby setting the stage favorably for the subsequent participation by CGA for their potential joint work in vesicle biogenesis. In this regard, the present results appear to underline the critical roles that CGB might play during the early stages of vesicle biogenesis and bring a new dimension to our perception of the potential functions accorded CGB.

  
Table: Effects of pH and Ca on chromogranin B conformation


  
Table: Comparison of conformations between chromogranin A and chromogranin B



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Neurochemistry, 5 Research Court, 2A37, NIDCD/NIH, Bethesda, MD 20892-3320. Tel.: 301-402-4223; Fax: 301-402-5475.

The abbreviations used are: CGA, chromogranin A; CGB, chromogranin B; bis-ANS, 4,4`-bis(1-anilinonaphthalene 8-sulfonate); MOPS, 3-(N-morpholino)propanesulfonic acid.

S. H. Yoo, manuscript in preparation.


ACKNOWLEDGEMENTS

I thank Dr. James Ferretti for use of the CD spectrometer.


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