C-Terminal Arginine-Selective Cleavage of Peptides as a Method for Mimicking Carboxypeptidase B.

C-Terminal residues play a pivotal role in dictating the structure and functions of proteins. Herein, we report a mild, efficient, chemoselective, and site-selective chemical method that allows for precise chemical proteolysis at C-terminal arginine dictated by 9,10-phenanthrenequinone independent of the remaining sequence. This biomimetic approach also exhibits the potential to synthesize C-terminal methyl ester (-CO2Me) peptides.

Limited proteolysis by a prostatic endopeptidase, the sperm-activating factor initiatorin, regulates the activation of pro-carboxypeptidase B in the seminal fluid of the silkworm, Bombyx mori.

A prostate trypsin-like serine endopeptidase called initiatorin (BmIni) is an essential factor in triggering the sperm maturation response of the silkworm, Bombyx mori. BmIni has been predicted to specifically cleave the carboxyl side of two consecutive arginine residues present in certain seminal plasma and sperm proteins, but the actual substrates are still unknown. In an attempt to elucidate the molecular mechanism underlying the sperm maturation signaling pathway, in this study, we examined whether BmIni activates the seminal carboxypeptidase B (BmCPB) protein through specific degradation. First, we confirmed in vitro that the inactive BmCPB present in unmated male vesicula (v.) seminalis is activated by treatment with BmIni or trypsin. Molecular cloning of the gene encoding the seminal BmCPB protein has shown that BmCPB is produced as a secreted proenzyme and may be activated after a trypsin-like protease cleaves the boundary between the prodomain and the enzyme site. In support of these findings, both trypsin and BmIni significantly activated recombinant Pro-BmCPB, which was successfully expressed and purified as a proenzyme in Escherichia coli; moreover, two specific cleavage forms appeared in the activation by BmIni that did not appear in that by trypsin. Therefore, a recombinant protein with a mutated diarginine motif (Arg109-Arg110), which is presumed to be a pre-cleavage site of BmCPB based on its high homology with bovine CPB, was prepared and treated with BmIni. As a result, the two specific degraded peptides were no longer observed, and simultaneously the activation was suppressed. Taken together, these findings lead to the conclusion that zymogen BmCPB, which is synthesized and secreted in male reproductive organs, is activated by sequence-dependent proteolysis by BmIni during ejaculation and in the female reproductive organs, providing a clue to the mechanism underlying seminal plasma and/or sperm protein degradation by BmIni in the sperm maturation cascade of B. mori.

Malaria Box Compounds against Anopheles gambiae (Diptera: Culicidae) Carboxypeptidase B Activity to Block Malaria Transmission.

Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl ß-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC50 at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.

Structure of Aedes aegypti procarboxypeptidase B1 and its binding with Dengue virus for controlling infection.

Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239 Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64-Glu84, Val238-Val252, and Leu278-Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.

Structure of Aedes aegypti carboxypeptidase B1-inhibitor complex uncover the disparity between mosquito and non-mosquito insect carboxypeptidase inhibition mechanism.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki  = 14.7 nM) is marginally less than that of bovine CPA1 (Ki  = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.

Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients.

BACKGROUND: Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. METHODS: We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay. RESULTS: We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. CONCLUSIONS: Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

Pancreatic Tissue Proteomics Unveils Key Proteins, Pathways, and Networks Associated with Type 1 Diabetes.

PURPOSE: Type 1 diabetes (T1D) is characterized by autoimmune mediated self-destruction of the pancreatic islet beta cells and the resultant insulin deficiency. However, little is known about the underlying molecular pathogenesis at the pancreatic tissue level given the limited availability of clinical specimens. EXPERIMENTAL DESIGN: Quantitative proteomic studies is performed on age-matched T1D and healthy cadaveric pancreatic tissues (n = 18 each) using TMT 10plex-based isobaric labeling and BoxCar-based label-free LC-MS/MS approaches. ELISA is used to validate the differentially expressed proteins (DEPs). RESULTS: Overall, the two quantitative proteomics approaches identified 8824 proteins, of which 261 are DEPs. KEGG pathway and functional network analyses of the DEPs reveal dysregulations to pancreatic exocrine function, complement coagulation cascades, and extracellular matrix receptor interaction pathways in T1D. A selected list of the DEPs associated with pathways, subnetworks, and plasma proteome of T1D are validated using ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: Integrating labeling and label-free approaches improve the confidence in quantitative profiling of pancreatic tissue proteome, which furthers the understanding of the dysregulated pathways and functional subnetworks associated with T1D pathogenesis and may aid to develop diagnostic and therapeutic strategies for T1D.

An 18O-Labeling Assisted LC-MS Method for Accurate Quantitation of Unprocessed C-Terminal Lysine in Therapeutic Monoclonal Antibodies.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

Carboxypeptidase B-Assisted Charge-Based Fractional Diagonal Chromatography for Deep Screening of C-Terminome.

The determination of protein C-termini is of great significance for protein function annotation and proteolysis research. However, the progress of C-terminomics is still far behind its counterpart, N-terminomics, because of the low reactivity of the carboxyl group. Herein, we developed a negative selection strategy, termed carboxypeptidase B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC), to achieve a global C-terminome analysis. The highly reactive carboxypeptidase B cleavage was utilized to reduce the charge state of non-C-terminal peptides. Together with high-performance charge-based fractional diagonal chromatography, the C-terminal peptides could be isolated. Such a strategy was applied for profiling C-termini from Escherichia coli cell lysates and 441 canonical C-termini and 510 neo-C-termini originating from proteolytic processing were identified. These findings represent 2-fold and 5.8-fold that of identified C-termini via direct analysis, respectively. Using parallel digestion with trypsin and LysC, such a strategy enabled the identification of 604 canonical C-termini and 818 neo-C-termini, representing the largest C-terminome data set of E. coli, and no deficiency in His/Lys/Arg-containing C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy is therefore a highly efficient and unbiased strategy for large-scale C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy, we identified 107 cleavage sites and 102 substrates of caspase-3 in Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows great promise in promoting proteolysis research. Data are available via ProteomeXchange with identifier PXD018520.

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2 . In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

Predictive Value of Novel Inflammation-Based Biomarkers for Pulmonary Hypertension in the Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Recently, there has been an increasing interest in the potential clinical use of several inflammatory indexes, namely, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic-immune-inflammation index (SII). This study aimed at assessing whether these markers could be early indicators of pulmonary hypertension (PH) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). A total of 185 patients were enrolled in our retrospective study from January 2017 to January 2019. Receiver operating characteristic curve (ROC) and area under the curve (AUC) were used to evaluate the clinical significance of these biomarkers to predict PH in patients with AECOPD. According to the diagnostic criterion for PH by Doppler echocardiography, the patients were stratified into two groups. The study group consisted of 101 patients complicated with PH, and the control group had 84 patients. The NLR, PLR, and SII values of the PH group were significantly higher than those of the AECOPD one (p < 0.05). The blood biomarker levels were positively correlated with NT-proBNP levels, while they had no significant correlation with the estimated pulmonary arterial systolic pressure (PASP) other than PLR. NLR, PLR, and SII values were all associated with PH (p < 0.05) in the univariate analysis, but not in the multivariate analysis. The AUC of NLR used for predicting PH was 0.701 and was higher than PLR and SII. Using 4.659 as the cut-off value of NLR, the sensitivity was 81.2%, and the specificity was 59.5%. In conclusion, these simple markers may be useful in the prediction of PH in patients with AECOPD.

Peptide-modified nanochannel system for carboxypeptidase B activity detection.

Carboxypeptidase B (CPB) is a protease that specifically hydrolyzes C-terminal alkaline amino acid of a peptide, which plays an important role in biological analysis. The activity and inhibition of CPB are significant for peptide sequencing and protein engineering. In this paper, a sensitive and easily-prepared nanochannel system was used to detect the activity of CPB. We assembled the peptides composing of alkaline amino acids into the nanochannels to detect the activity of CPB based on its hydrolysis characteristic. With CPB, the peptides would be cleaved, causing less blocking-effect on the ionic current through nanochannels. This system exhibited high sensitivity (detection limit of 0.01 U mL-1), wide linear range (0.01-10 U mL-1), and fast response (less than 10 s) for specific CPB detection. We also used the system to detect the effect of CPB inhibitors and detect in complex actual samples. The strategy exhibits effective analytical characteristics and it can be regarded as a hopeful prospect for the rapid diagnosis of patients with pancreatitis.

Crystal structure of mutant carboxypeptidase T from Thermoactinomyces vulgaris with an implanted S1' subsite from pancreatic carboxypeptidase B.

A site-directed mutagenesis method has been used to obtain the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT), in which the amino-acid residues of the S1' subsite are substituted by the corresponding residues from pancreatic carboxypeptidase B (CPB). It was shown that the mutant enzyme retained the broad, mainly hydrophobic selectivity of wild-type CPT. The mutant containing the implanted CPB S1' subsite was crystallized and its three-dimensional structure was determined at 1.29 Šresolution by X-ray crystallography. A comparison of the three-dimensional structures of CPT, the G215S/A251G/T257A/D260G/T262D CPT mutant and CPB showed that the S1' subsite of CPT has not been distorted by the mutagenesis and adequately reproduces the structure of the CPB S1' subsite. The CPB-like mutant differs from CPB in substrate selectivity owing to differences between the two enzymes outside the S1' subsite. Moreover, the difference in substrate specificity between the enzymes was shown to be affected by residues other than those that directly contact the substrate.

Mutations in the pancreatic secretory enzymes CPA1 and CPB1 are associated with pancreatic cancer.

To evaluate whether germline variants in genes encoding pancreatic secretory enzymes contribute to pancreatic cancer susceptibility, we sequenced the coding regions of CPB1 and other genes encoding pancreatic secretory enzymes and known pancreatitis susceptibility genes (PRSS1, CPA1, CTRC, and SPINK1) in a hospital series of pancreatic cancer cases and controls. Variants in CPB1, CPA1 (encoding carboxypeptidase B1 and A1), and CTRC were evaluated in a second set of cases with familial pancreatic cancer and controls. More deleterious CPB1 variants, defined as having impaired protein secretion and induction of endoplasmic reticulum (ER) stress in transfected HEK 293T cells, were found in the hospital series of pancreatic cancer cases (5/986, 0.5%) than in controls (0/1,045, P = 0.027). Among familial pancreatic cancer cases, ER stress-inducing CPB1 variants were found in 4 of 593 (0.67%) vs. 0 of 967 additional controls (P = 0.020), with a combined prevalence in pancreatic cancer cases of 9/1,579 vs. 0/2,012 controls (P < 0.01). More ER stress-inducing CPA1 variants were also found in the combined set of hospital and familial cases with pancreatic cancer than in controls [7/1,546 vs. 1/2,012; P = 0.025; odds ratio, 9.36 (95% CI, 1.15-76.02)]. Overall, 16 (1%) of 1,579 pancreatic cancer cases had an ER stress-inducing CPA1 or CPB1 variant, compared with 1 of 2,068 controls (P < 0.00001). No other candidate genes had statistically significant differences in variant prevalence between cases and controls. Our study indicates ER stress-inducing variants in CPB1 and CPA1 are associated with pancreatic cancer susceptibility and implicate ER stress in pancreatic acinar cells in pancreatic cancer development.

Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Phosphorylation of carboxypeptidase B1 protein regulates ß-cell proliferation.

A reduction in pancreatic islet ß-cells leads to the onset of diabetes. Hence, the identification of the mechanisms inducing ß-cell proliferation is important for developing a treatment course against the disease. It has been well established that post-translational modifications (PTMs) of proteins affect their functionality. In addition, PTMs have been suggested to play important roles in organ regeneration. Therefore, in this study, we investigated PTMs associated with pancreatic regeneration using two-dimensional electrophoresis. Four carboxypeptidase B1 (CPB1) proteins were identified at different isoelectric points, with the same molecular weight. The motif of CPB1 PTMs was identified by mass spectrophotometry, and the downregulation of CPB1 phosphorylation in pancreatectomy was confirmed. The dephosphorylation of CPB1 induced ß-cell proliferation. We thus surmise that the altered PTM of CPB1 is associated with pancreatic regeneration.

Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity.

Cell death and release of proinflammatory mediators contribute to mortality during sepsis. Specifically, caspase-11-dependent cell death contributes to pathology and decreases in survival time in sepsis models. Priming of the host cell, through TLR4 and interferon receptors, induces caspase-11 expression, and cytosolic LPS directly stimulates caspase-11 activation, promoting the release of proinflammatory cytokines through pyroptosis and caspase-1 activation. Using a CRISPR-Cas9-mediated genome-wide screen, we identified novel mediators of caspase-11-dependent cell death. We found a complement-related peptidase, carboxypeptidase B1 (Cpb1), to be required for caspase-11 gene expression and subsequent caspase-11-dependent cell death. Cpb1 modifies a cleavage product of C3, which binds to and activates C3aR, and then modulates innate immune signaling. We find the Cpb1-C3-C3aR pathway induces caspase-11 expression through amplification of MAPK activity downstream of TLR4 and Ifnar activation, and mediates severity of LPS-induced sepsis (endotoxemia) and disease outcome in mice. We show C3aR is required for up-regulation of caspase-11 orthologues, caspase-4 and -5, in primary human macrophages during inflammation and that c3aR1 and caspase-5 transcripts are highly expressed in patients with severe sepsis; thus, suggesting that these pathways are important in human sepsis. Our results highlight a novel role for complement and the Cpb1-C3-C3aR pathway in proinflammatory signaling, caspase-11 cell death, and sepsis severity.

Complex Formation of Human Proelastases with Procarboxypeptidases A1 and A2.

The pancreas secretes digestive proenzymes typically in their monomeric form. A notable exception is the ternary complex formed by proproteinase E, chymotrypsinogen C, and procarboxypeptidase A (proCPA) in cattle and other ruminants. In the human and pig pancreas binary complexes of proCPA with proelastases were found. To characterize complex formation among human pancreatic protease zymogens in a systematic manner, we performed binding experiments using recombinant proelastases CELA2A, CELA3A, and CELA3B; chymotrypsinogens CTRB1, CTRB2, CTRC, and CTRL1; and procarboxypeptidases CPA1, CPA2, and CPB1. We found that proCELA3B bound not only to proCPA1 (KD 43 nm) but even more tightly to proCPA2 (KD 18 nm), whereas proCELA2A bound weakly to proCPA1 only (KD 152 nm). Surprisingly, proCELA3A, which shares 92% identity with proCELA3B, did not form stable complexes due to the evolutionary replacement of Ala(241) with Gly. The polymorphic nature of position 241 in both CELA3A (∼4% Ala(241) alleles) and CELA3B (∼2% Gly(241) alleles) points to individual variations in complex formation. The functional effect of complex formation was delayed procarboxypeptidase activation due to increased affinity of the inhibitory activation peptide, whereas proelastase activation was unchanged. We conclude that complex formation among human pancreatic protease zymogens is limited to a subset of proelastases and procarboxypeptidases. Complex formation stabilizes the inhibitory activation peptide of procarboxypeptidases and thereby increases zymogen stability and controls activation.

Effects of Rituximab and Infliximab Treatment on Carboxypeptidase B and Its Substrates in RA Synovium.

OBJECTIVE: We evaluated the synovial effects of 2 potent biologic rheumatoid arthritis (RA) therapies, focusing on their effect on the expression level of carboxypeptidase B (CPB) and its substrates. METHODS: Patients with RA receiving infliximab (IFX; n = 9) or rituximab (RTX; n = 5) had an arthroscopic synovial biopsy at baseline and 16 weeks posttherapy. Expression of CPB, C5a, osteopontin (OPN), CD3, CD20, CD55, and CD68 was assessed by immunohistochemistry and image analysis, and compared with OA synovium. RA disease activity score was assessed at multiple timepoints. Serial serum samples were analyzed for soluble CPB and C5a levels. RESULTS: The baseline clinical characteristics of patients receiving IFX and RTX were similar. At the time of the second biopsy, 50% of patients had achieved a European League Against Rheumatism good or moderate response. At baseline, expression of CPB, C5a, and OPN was markedly higher in RA compared with OA synovium and correlated with mononuclear cell infiltration. There was an overall reduction in synovial expression of CPB, C5a, and OPN paralleling a reduction in mononuclear cell infiltration, but these changes were not associated with clinical response. After an early reduction in serum C5a levels, these returned to baseline levels at later timepoints. CONCLUSION: In response to IFX and RTX treatment, RA synovial expression of CPB, C5a, and OPN decrease independently of the clinical response, reflecting the complex proinflammatory and antiinflammatory effects of this pathway.

Variants in pancreatic carboxypeptidase genes CPA2 and CPB1 are not associated with chronic pancreatitis.

Genetic alterations in the carboxypeptidase A1 gene (CPA1) are associated with early onset chronic pancreatitis (CP). Besides CPA1, there are two other human pancreatic carboxypeptidases (CPA2 and CPB1). Here we examined whether CPA2 and CPB1 alterations are associated with CP in Japan and Germany. All exons and flanking introns of CPA2 and CPB1 were sequenced in 477 Japanese patients with CP (234 alcoholic, 243 nonalcoholic) and in 497 German patients with nonalcoholic CP by targeted next-generation sequencing and/or Sanger sequencing. Secretion and enzymatic activity of CPA2 and CPB1 variants were determined after transfection into HEK 293T cells. We identified six nonsynonymous CPA2 variants (p.V67I, p.G166R, p.D168E, p.D173H, p.R237W, and p.G388S), eight nonsynonymous CPB1 alterations (p.S65G, p.N120S, p.D172E, p.R195H, p.D208N, p.F232L, p.A317V, and p.D364Y), and one splice-site variant (c.687+1G>T) in CPB1. Functional analysis revealed essentially complete loss of function in CPA2 variants p.R237W and p.G388S and CPB1 variants p.R110H and p.D364Y. None of the CPA2 or CPB1 variants, including those resulting in a marked loss of function, were overrepresented in patients with CP. In conclusion, CPA2 and CPB1 variants are not associated with CP.