Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

Wang, Haibo; Yuan, Duo-Duo; Xu, Rong; Wu, Cheng

doi:10.1093/abbs/gmr047

Cited by 6 publications

(4 citation statements)

References 19 publications

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“…Chymotrypsin C (CTRC) is a digestive serine protease secreted by the acinar cells of the pancreas as an inactive zymogen (chymotrypsinogen C) which becomes activated in the duodenum after trypsin‐mediated cleavage of the activation peptide [1]. The activation peptide remains attached to the enzyme through the Cys17‐Cys141 disulfide link and may be further processed by CTRC to a shorter form [2]. CTRC cleaves dietary proteins and peptides after aromatic (Phe, Tyr) and aliphatic (Leu) amino acid residues with characteristically high activity on leucyl peptide bonds [3–6].…”

Section: Introductionmentioning

confidence: 99%

Asparagine‐linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Bence

Sahin–Tóth

2011

The FEBS Journal

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SUMMARY Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion, however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.

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Section: Introductionmentioning

confidence: 99%

Asparagine‐linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Bence

Sahin–Tóth

2011

The FEBS Journal

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“…The geodetector is a statistical tool that detects the spatial heterogeneity of an element and reveals the driving force behind it (Wang and Xu, 2017). The theoretical core of the detector is to detect the consistency of the dependent variable and the independent variable through spatial heterogeneity.…”

Section: Driving Force Analysis Of Ecosystem Servicesmentioning

confidence: 99%

Profoundly entwined ecosystem services, land-use change and human well-being into sustainability management in Yushu, Qinghai-Tibet Plateau

et al. 2022

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The nexus exploration among land use/land cover change, ecosystem services and human well-being has been increasingly crucial in the context of Future Earth. However, the spatial heterogeneity and the entwining process among these three aspects have not yet been in-depth and systematically explored. Here we identified the spatiotemporal pattern of ecosystem services during the past 20 years in Yushu, the eco-fragile region and the centre of Qinghai-Tibet Plateau, as well as clarified its relationships with land use change and human well-being. We revealed that: (1) The structure of the ecosystem and land use in this area have been increasingly stable, and the ecological projects have exerted a positive impact. (2) Although the ecological environmental issues still need more attention, the ecosystem services of the area have been positively developing. (3) Derived by the ecosystem services increase, environmental projects and policies, the human well-beings of culture and education performed much better than other aspects. (4) It is crucial to carry out long-term ecological projects and increase educational investment for maintaining the stability of this ecologically fragile area. This study provides significant support for the regional ecological sustainability decision making, especially for the Qinghai-Tibet Plateau, the roof of the world.

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“…Chymotrypsin C possesses two barrel structures, between which the charge-relayed catalytic triad (His74, Asp121, and Ser216) is located. The activation peptide is first cleaved at the Arg29–Ile30 peptide bond by trypsin, and further cleaved at Asp25–Leu26 [ 10 ] or Leu26–Ser27 [ 19 ] by the autoactivation of chymotrypsin C. The cleaved Cys17–Asp25 or Cys17–Leu26 long peptide remains attached to the mature protein by a disulfide bridge such as Cys17–Cys141, a structure that resembles chymotrypsin [ 10 , 14 , 15 , 19 , 20 ].…”

Section: Protein Structure and Protease Activity Of Caldecrinmentioning

confidence: 99%

Purification and Biological Function of Caldecrin

2021

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Blood calcium homeostasis is critical for biological function. Caldecrin, or chymotrypsin-like elastase, was originally identified in the pancreas as a serum calcium-decreasing factor. The serum calcium-decreasing activity of caldecrin requires the trypsin-mediated activation of the protein. Protease activity-deficient mature caldecrin can also reduce serum calcium concentration, indicating that structural processing is necessary for serum calcium-decreasing activity. Caldecrin suppresses the differentiation of bone-resorbing osteoclasts from bone marrow macrophages (BMMs) by inhibiting receptor activator of NF-κB ligand (RANKL)-induced nuclear factor of activated T-cell cytoplasmic 1 expression via the Syk–PLCγ–Ca2+ oscillation-calcineurin signaling pathway. It also suppresses mature osteoclastic bone resorption by RANKL-stimulated TRAF6–c-Src–Syk–calcium entry and actin ring formation. Caldecrin inhibits lipopolysaccharide (LPS)-induced osteoclast formation in RANKL-primed BMMs by inducing the NF-κB negative regulator A20. In addition, caldecrin suppresses LPS-mediated M1 macrophage polarization through the immunoreceptor triggering receptor expressed on myeloid cells (TREM) 2, suggesting that caldecrin may function as an anti-osteoclastogenic and anti-inflammatory factor via TREM2. The ectopic intramuscular expression of caldecrin cDNA prevents bone resorption in ovariectomized mice, and the administration of caldecrin protein also prevents skeletal muscle destruction in dystrophic mice. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease and a possible therapeutic target for skeletal and inflammatory diseases.

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Purification, cDNA cloning, and recombinant expression of chymotrypsin C from porcine pancreas

Cited by 6 publications

References 19 publications

Asparagine‐linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Asparagine‐linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Profoundly entwined ecosystem services, land-use change and human well-being into sustainability management in Yushu, Qinghai-Tibet Plateau

Purification and Biological Function of Caldecrin

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