Two human trypsinogens. Purification, molecular properties, and N-terminal sequences

Guy, Odette; Lagadic‐Gossmann, Dominique; Bartelt, Diana C.; Amic, J; Figarella, Catherine

doi:10.1021/bi00602a014

Cited by 120 publications

(56 citation statements)

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“…In any case, the sequence obtained allows this protein to be assigned unequivocably as human proproteinase E, since it fits the N-terminal end of the same protein exactly, as recently deduced from its cDNA sequence [33]. This sequence is also different from that reported for the equivalent region in other serine proproteinases from humans [35] or other mammals [33].…”

Section: Hplc Purijkation Ojhuman Procarboxypeptidasesmentioning

confidence: 79%

Purification and properties of five different forms of human procarboxypeptidases

Pascual

Burgos

Salvà

et al. 1989

European Journal of Biochemistry

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Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A3 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.The existence of procarboxypeptidases in two forms which generate active enzymes of different specificity, the A and the B forms, is a widely recognized fact [l]. However, the presence of further isology in these forms has been studied in depth in only a few species. Thus, Neurath et al. [2, 31 have isolated and characterized two alellomorphic forms of bovine carboxypeptidase A. Both forms were isolated under autolytic conditions and no information was obtained on the possible existence of further heterogeneity in the precursor molecules. Scheele et al. [4-81 have also obtained evidence, using electrophoretic techniques, for the existence of two or three forms of procarboxypeptidase A protomer and one to three forms of procarboxypeptidase B in different mammals (guinea pig, rat, dog and human). Recently, Rutter and coworkers [9, 101 have reported the probable existence of a single procarboxypeptidase A gene in the rat genome and have already located the chromosome bands that contain the gene(s) for the same protein in humans and mice.This work reports, for the first time, the rapid and complete isolation of different homologous procarboxypeptidases, three A forms and two B forms, in a native state from human pancreas, using HPLC. The existence of a stable binary complex of a procarboxypeptidase A and a proproteinase E is also reported. Several fundamental properties of these proteins have been characterized and compared with those of the equivalent proenzymes from porcine pancreas, which have been previously studied by our group [11 -181. In addition, the chromatographic behaviour of these human procarboxypeptidases has been analyzed under different conditions and using different HPL...

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Section: Hplc Purijkation Ojhuman Procarboxypeptidasesmentioning

confidence: 79%

Purification and properties of five different forms of human procarboxypeptidases

Pascual

Burgos

Salvà

et al. 1989

European Journal of Biochemistry

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“…The isoenzymes are encoded by separate genes, the PRSS1 (protease, serine, 1), PRSS2 and PRSS3 genes (for a recent review see [1] and references therein). Cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) make up the bulk of secreted trypsinogens in the pancreatic juice, while mesotrypsinogen (PRSS3) accounts for 2-10 % [2][3][4][5][6]. Typically, there is approximately twice as much cationic trypsinogen as anionic trypsinogen, but this ratio is reversed in chronic alcoholism or chronic pancreatitis [3,5].…”

Section: Human Trypsinogens and Pancreatitis-associated Trypsinogen Mmentioning

confidence: 99%

Biochemical Models of Hereditary Pancreatitis

Sahin–Tóth

2006

Endocrinology and Metabolism Clinics of North America

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The past decade has witnessed remarkable progress in the genetics of chronic pancreatitis. Mutations of the PRSS1 gene encoding cationic trypsinogen and the SPINK1 gene encoding pancreatic secretory trypsin inhibitor were found in association with hereditary, familial or sporadic chronic pancreatitis; and the genotype -phenotype correlations have been characterized at the clinical level. Despite these accomplishments, our understanding of the molecular mechanism(s) through which PRSS1 and SPINK1 mutations cause chronic pancreatitis has remained sketchy. Pancreatitis-associated gene mutations are believed to result in uncontrolled trypsin activity in the pancreas. Thus, PRSS1 mutations would cause a gain of (trypsin) function, while SPINK1 mutations would result in the loss of a (trypsin inhibitor) function. However, experimental identification of the disease-relevant functional alterations caused by PRSS1 or SPINK1 mutations proved to be challenging, as results of biochemical analyses lent themselves to different interpretations. The present review focuses on PRSS1 mutations and summarizes the salient biochemical findings in the context of the mechanistic models that attempt to explain the connection between mutations and hereditary pancreatitis. Human Trypsinogens and Pancreatitis-Associated Trypsinogen MutationsThe human pancreas produces the digestive pro-enzyme trypsinogen in three isoforms. On the basis of their relative isoelectric points and electrophoretic mobility, these are commonly referred to as cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen. The isoenzymes are encoded by separate genes, the PRSS1 (protease, serine, 1), PRSS2 and PRSS3 genes (for a recent review see [1] and references therein). Cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2) make up the bulk of secreted trypsinogens in the pancreatic juice, while mesotrypsinogen (PRSS3) accounts for 2-10 % [2-6]. Typically, there is approximately twice as much cationic trypsinogen as anionic trypsinogen, but this ratio is reversed in chronic alcoholism or chronic pancreatitis [3,5]. The significance of the "isoform reversal" is unknown [7]. Human trypsinogens are synthesized as pre-pro-enzymes with a signal peptide of 15 amino acids, followed by the 8 amino acid long pro-peptide, the trypsinogen activation peptide. The signal-peptide is removed upon entry into the endoplasmic reticulum lumen and the proenzymes are packaged into zymogen granules and eventually secreted into the pancreatic juice. Physiological activation of trypsinogen to trypsin takes place in the duodenum by enteropeptidase (enterokinase), a highly specialized serine protease in the brush-border membrane of enterocytes. Trypsin can also activate trypsinogen, a process termed autoactivation, which in the duodenum may have a physiological role in facilitating zymogen activation, whereas inappropriate autoactivation in the pancreas might cause pancreatitis. Mutations in the PRSS1 gene have been identified in patients with hereditary pancreatitis, fam...

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“…The human pancreas secretes three isoforms as follows: cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen, which are encoded by the PRSS1, PRSS2, and PRSS3 genes, respectively (1). Cationic and anionic trypsinogen constitute more than 95% of total trypsinogen in the pancreatic juice, with a slight preponderance of the cationic isoform (2)(3)(4). Interestingly, expression of anionic trypsinogen is increased in inflammatory and malignant diseases of the pancreas and in chronic alcoholism resulting in a reversal of the relative isoform amounts (4 -6).…”

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confidence: 99%

“…Hereditary pancreatitis is an autosomal dominant disorder caused by mutations in PRSS1 that result in increased intra-pancreatic trypsinogen activation (8,9). The mutations alter cleavage of regulatory nick sites by chymotrypsin C (CTRC) 2 and trypsin that control trypsinogen degradation and autoactivation (10).…”

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confidence: 99%

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

Jancsó

Sahin–Tóth

2016

Journal of Biological Chemistry

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The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139 -Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

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Two human trypsinogens. Purification, molecular properties, and N-terminal sequences

Cited by 120 publications

References 41 publications

Purification and properties of five different forms of human procarboxypeptidases

Purification and properties of five different forms of human procarboxypeptidases

Biochemical Models of Hereditary Pancreatitis

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

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