Ionic interactions between bovine chymotrypsinogen A and chondroitin sulfate A.B.C.. A possible model for molecular aggregation in zymogen granules.

Abstract

The formation of large aggregates by ionic interactions between acidic glucosaminoglycans and cationic secretory proteins has been proposed as one of the critical steps in the concentration process in the condensing vacuoles of secretory cells. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A and chondroitin sulfate as a simplified model. Small amounts of chondroitin sulfate were found able to induce chymotrypsinogen precipitation. Like zymogen granules, the resulting aggregates were moderately sensitive to ionic strength and insensitive to osmolality. Moreover, their pH dependence was similar to that of isolated zymogen granules. When sulfated glucosaminoglycans isolated from the zymogen granules of the guinea pig pancreas were used instead of chondroitin sulfate, the same kind of interactions with chymotrypsinogen were obtained. Our data support the hypothesis that the strong ionic inteFactions between those sulfated glucosaminoglycans and cationic proteins could be responsible for the concentration process.KEY WORDS chymotrypsinogen A 9 chondroitin sulfate glucosaminoglycans molecular aggregation zymogen granule condensation turbidity A sulfated macromolecular fraction has recently been isolated from the zymogen granules of the guinea pig pancreas (17, 18). Preliminary results indicate that this fraction is composed of acidic glucosaminoglycans, principally heparan sulfate and chondroitin sulfate (18). Such compounds have been implicated in the process by which pancreatic acinar cells concentrate their secretory proteins in the condensing vacuoles of the Golgi complex (23,16,9). The formation of large aggregates by ionic interactions between those polyanions and cationic secretory proteins could reduce the osmotic activity within the vacuoles and lead to concentration of their content by water loss. In this paper, this hypothesis was tested by studies on the interactions between bovine chymotrypsinogen A (ChTg) and chondroitin sulfate (Ch-SO4) as a simplified model. The results indicate that the ionic interactions of these compounds are strong enough to induce coprecipitation. Sulfated polyanions isolated from the zymogen granules of the guinea pig pancreas were found to interact with bovine ChTg in a manner comparable to that of Ch-SO4. MATERIALS AND METHODSBovine chymotrypsinogen A (Worthington Biochemical Corp., Freehold, N.J.) dissolved (1 mg/ml) in 2 mM Tris maleate buffer, pH 6, was mixed with solutions of J. CELL BIOLOGY 9 The Rockefeller University Press -0021-9525/78/0901-095151.00 951 on

Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

“…Similarly, a sulfated glycoprotein plays a role in protein storage in pancreatic exocrine cells (De Lisle, 2002). Indeed, the aggregation of regulated secretory proteins and glycosaminoglycans was first described for exocrine pancreatic secretory granules and was proposed to play a role in protein storage (Reggio and Dagorn, 1978). Unlike mast and exocrine cells, sulfated proteoglycans are not necessary for protein storage in secretory granules of pituitary AtT-20 cells (Burgess and Kelly, 1984) or pituitary GH4C 1 cells (Gorr, 1996).…”

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Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

“…Conceivably, conserved structural features might allow for interaction with a conserved transGolgi network (TGN)-based sorting receptor (Chung et al, 1989) directing protein entry into immature granules (IGs), which is the first compartment in the regulated secretory pathway to acquire competence for stimulus-dependent release (Arvan et al, 1991;Tooze et al, 1991;Carnell and Moore, 1994). In addition, assembly of insoluble complexes, a common feature among regulated secretory proteins (Giannattasio et al, 1975;Reggio and Dagorn, 1978;Chanat and Huttner, 1991), leads to granule core formation within the intraluminal environment (Verbsky and Turkewitz, 1998) that may facilitate the ultimate targeting of regulated secretory proteins (Kuliawat and Arvan, 1992). Thus, after oligomerization in the early secretory pathway (Huang and Arvan, 1995;Thiele and Huttner, 1998a), the development of a higher-order quaternary structure within the distal secretory pathway may play a significant role in the intragranular storage of regulated secretory proteins (Thiele et al, 1997;Arvan and Castle, 1998).…”

Proteoglycans support proper granule formation in pancreatic acinar cells

“…For the exocrine pancreas, however, studies showing the presence of proteoglycans in ZGs have so far been descriptive only. In vitro studies suggested that sulfated proteoglycans within ZGs might mediate the sorting and aggregation of cationic zymogens via ionic interactions of their negatively charged sulfate groups (Reggio & Dagorn 1978, Reggio & Palade 1978, Tartakoff et al 1975. Using the CmB protocol as well as gel-based analyses, we provided direct morphological and biochemical evidence for the presence of proteoglycans within the ZGs of rat pancreas.…”