Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

1989 ◽  
Vol 67 (6) ◽  
pp. 281-287 ◽  
Author(s):  
Denis Lebel ◽  
Jean Paquette
Keyword(s):  
Disulfide Bonds ◽  
Hydrophobic Interaction Chromatography ◽  
Zymogen Granule ◽  
Proteolytic Digestion ◽  
Tryptic Fragment ◽  
Reducing Conditions ◽  
Granule Membrane ◽  
Reconstituted System ◽  
Specific Degradation

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.Key words: GP-2, zymogen granule, disulfide, exocrine, pancreas, secretion.

Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms

1986 ◽  
Vol 64 (5) ◽  
pp. 456-462 ◽  
Author(s):  
Jean Paquette ◽  
François A. Leblond ◽  
Marlyne Beattie ◽  
Denis LeBel
Keyword(s):  
Membrane Protein ◽  
Gel Filtration ◽  
Soluble Form ◽  
Major Protein ◽  
Zymogen Granule ◽  
Chloromethyl Ketone ◽  
Reducing Conditions ◽  
Immunochemical Quantitation ◽  
Granule Membrane ◽  
Different Levels

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-α-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.

scholarly journals Stimulation of exocytotic membrane fusion by modified peptides of the rab3 effector domain: re-evaluation of the role of rab3 in regulated exocytosis

1993 ◽  
Vol 294 (2) ◽  
pp. 325-328 ◽  
Author(s):  
C M MacLean ◽  
G J Law ◽  
J M Edwardson
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Fusion Event ◽  
Effector Domain ◽  
Granule Membrane ◽  
Modified Peptides ◽  
Stimulation Of

We have shown previously that fusion between pancreatic zymogen granules and plasma membranes is stimulated by a peptide corresponding to the putative effector domain of rab3. Here we show that this stimulatory effect persists when the amino acid sequence of the peptide is substantially modified. We also show that an antibody raised against rab3a recognizes a protein of appropriate size on the zymogen-granule membrane, but has no effect on membrane fusion. We suggest that rab3 is not directly involved in the control of this membrane fusion event, and that the peptides are stimulating fusion by a mechanism unrelated to rab3.

scholarly journals Syncollin is required for efficient zymogen granule exocytosis

2005 ◽  
Vol 385 (3) ◽  
pp. 721-727 ◽  
Author(s):  
Barbara WÄSLE ◽  
Matthew TURVEY ◽  
Olga LARINA ◽  
Peter THORN ◽  
Jeremy SKEPPER ◽  
...  
Keyword(s):  
Physiological Role ◽  
Pancreatic Acinar Cell ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Wild Type ◽  
Amylase Release ◽  
Two Photon ◽  
Compound Exocytosis ◽  
Granule Membrane

Syncollin is a 13 kDa protein that is present in the exocrine pancreas, where the majority of the protein is tightly attached to the luminal surface of the zymogen granule membrane. We have addressed the physiological role of syncollin by studying the phenotype of syncollin KO (knockout) mice. These mice show pancreatic hypertrophy and elevated pancreatic amylase levels. Further, secretagogue-stimulated amylase release from pancreatic lobules of syncollin KO mice was found to be reduced by about 45% compared with wild-type lobules, and the delivery of newly synthesized protein to zymogen granules was delayed, indicating that the mice have a pancreatic secretory defect. As determined by two-photon imaging, the number of secretagogue-stimulated exocytotic events in acini from syncollin KO mice was reduced by 50%. This reduction was accounted for predominantly by a loss of later, ‘secondary’ fusion events between zymogen granules and other granules that had already fused with the plasma membrane. We conclude that syncollin is required for efficient exocytosis in the pancreatic acinar cell, and that it plays a particularly important role in compound exocytosis.

scholarly journals Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A

1997 ◽  
Vol 324 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Nigel M. HOOPER ◽  
Sarah COOK ◽  
Jean LAINÉ ◽  
Denis LeBEL
Keyword(s):  
Enzyme Activity ◽  
Phospholipase A ◽  
Zymogen Granule ◽  
Binding Properties ◽  
Glycosyl Phosphatidylinositol ◽  
Reducing Conditions ◽  
Sds Page ◽  
Granule Membrane ◽  
Membrane Bound ◽  
Polyclonal Antisera

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin–Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.

Folding of peptides and proteins: role of disulfide bonds, recent developments

2013 ◽  
Vol 4 (6) ◽  
pp. 597-604 ◽  
Author(s):  
Yuji Hidaka ◽  
Shigeru Shimamoto
Keyword(s):  
Protein Folding ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Difficult Problem ◽  
Chemical Methods ◽  
Mutant Proteins ◽  
Folding Intermediates ◽  
Peptide Chemistry ◽  
Recent Developments

AbstractDisulfide-containing proteins are ideal models for studies of protein folding as the folding intermediates can be observed, trapped, and separated by HPLC during the folding reaction. However, regulating or analyzing the structures of folding intermediates of peptides and proteins continues to be a difficult problem. Recently, the development of several techniques in peptide chemistry and biotechnology has resulted in the availability of some powerful tools for studying protein folding in the context of the structural analysis of native, mutant proteins, and folding intermediates. In this review, recent developments in the field of disulfide-coupled peptide and protein folding are discussed, from the viewpoint of chemical and biotechnological methods, such as analytical methods for the detection of disulfide pairings, chemical methods for disulfide bond formation between the defined Cys residues, and applications of diselenide bonds for the regulation of disulfide-coupled peptide and protein folding.

Role of disulfide bonds in folding and secretion of human lysozyme in saccharomycescerevisiae

1988 ◽  
Vol 152 (3) ◽  
pp. 962-967 ◽  
Author(s):  
Yoshio Taniyama ◽  
Yoshio Yamamoto ◽  
Masafumi Nakao ◽  
Masakazu Kikuchi ◽  
Morio Ikehara
Keyword(s):  
Disulfide Bonds ◽  
Human Lysozyme

scholarly journals Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110010 ◽  
Author(s):  
Clive Metcalfe ◽  
Peter Cresswell ◽  
Laura Ciaccia ◽  
Benjamin Thomas ◽  
A. Neil Barclay
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Disulfide Bonds ◽  
Reduction Process ◽  
Cysteine Residue ◽  
Surface Proteins ◽  
Protein Activity ◽  
Functional Changes ◽  
Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

scholarly journals NBR1 directly promotes the formation of p62 – ubiquitin condensates via its PB1 and UBA domains

2020 ◽  
Author(s):  
Adriana Savova ◽  
Julia Romanov ◽  
Sascha Martens
Keyword(s):  
Phase Separation ◽  
Wild Type ◽  
Selective Autophagy ◽  
Ubiquitinated Proteins ◽  
Cargo Receptor ◽  
Condensate Formation ◽  
Uba Domain ◽  
Reconstituted System ◽  
Pb1 Domain

SummarySelective autophagy removes harmful intracellular structures such as ubiquitinated, aggregated proteins ensuring cellular homeostasis. This is achieved by the encapsulation of this cargo material within autophagosomes. The cargo receptor p62/SQSTM1 mediates the phase separation of ubiquitinated proteins into condensates, which subsequently become targets for the autophagy machinery. NBR1, another cargo receptor, is a crucial regulator of condensate formation. The mechanisms of the interplay between p62 and NBR1 are not well understood. Employing a fully reconstituted system we show that two domains of NBR1, the PB1 domain which binds to p62 and the UBA domain which binds to ubiquitin, are required to promote p62-ubiquitin condensate formation. In cells, acute depletion of endogenous NBR1 reduces formation of p62 condensates, a phenotype that can be rescued by re-expression of wild-type NBR1, but not PB1 or UBA domain mutants. Our results provide mechanistic insights into the role of NBR1 in selective autophagy.

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