Human Single-Chain Antibodies That Neutralize Elastolytic Activity of Pseudomonas aeruginosa LasB

LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting Pseudomonas aeruginosa. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the P. aeruginosa. This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (huscfvs) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the huscfv-plasmid transformed Escherichia coli. HuscFvs of two transformed E. coli clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities in vitro. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by P. aeruginosa.

PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity

Background Obesity-related cardiovascular risk, end points, and mortality are strongly related to arterial stiffening. Current therapeutic approaches for arterial stiffening are not focused on direct targeting within the vessel. Perivascular adipose tissue (PVAT) surrounding the artery has been shown to modulate vascular function and inflammation. Peroxisome proliferator-activated receptor γ (PPARγ) activation significantly decreases arterial stiffness and inflammation in diabetic patients with coronary artery disease. Thus, we hypothesized that PPARγ activation alters the PVAT microenvironment, thereby creating a favorable environment for the attenuation of arterial stiffening in obesity. Methods Obese ob/ob mice were used to investigate the effect of PPARγ activation on the attenuation of arterial stiffening. Various cell types, including macrophages, fibroblasts, adipocytes, and vascular smooth muscle cells, were used to test the inhibitory effect of pioglitazone, a PPARγ agonist, on the expression of elastolytic enzymes. Results PPARγ activation by pioglitazone effectively attenuated arterial stiffening in ob/ob mice. This beneficial effect was not associated with the repartitioning of fat from or changes in the browning of the PVAT depot but was strongly related to improvement of the PVAT microenvironment, as evidenced by reduction in the expression of pro-inflammatory and pro-oxidative factors. Pioglitazone treatment attenuated obesity-induced elastin fiber fragmentation and elastolytic activity and ameliorated the obesity-induced upregulation of cathepsin S and metalloproteinase 12, predominantly in the PVAT. In vitro, pioglitazone downregulated Ctss and Mmp12 in macrophages, fibroblasts, and adipocytes—cell types residing within the adventitia and PVAT. Ultimately, several PPARγ binding sites were found in Ctss and Mmp12 in Raw 264.7 and 3T3-L1 cells, suggesting a direct regulatory mechanism by which PPARγ activation repressed the expression of Ctss and Mmp-12 in macrophages and fibroblasts. Conclusions PPARγ activation attenuated obesity-induced arterial stiffening and reduced the inflammatory and oxidative status of PVAT. The improvement of the PVAT microenvironment further contributed to the amelioration of elastin fiber fragmentation, elastolytic activity, and upregulated expression of Ctss and Mmp12. Our data highlight the PVAT microenvironment as an important target against arterial stiffening in obesity and provide a novel strategy for the potential clinical use of PPARγ agonists as a therapeutic against arterial stiffness through modulation of PVAT function.

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation

Imbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment.

Indications that Chymotrypsin-like Elastase 1 is Involved in Emphysema

ABSTRACTEmphysema is an important element of many progressive lung diseases, with chronic obstructive pulmonary disease (COPD) being the most common. With the exception of α 1-antitrypsin (AAT) replacement therapy there are no disease modifying therapies for progressive emphysema. We previously reported that alveolar type 2 (AT2)-cell synthesized CELA1 is neutralized by AAT and that CELA1 is necessary for emphysema in AAT-deficiency. Here, we use mouse models and human tissues to show that CELA1 is required for progressive emphysema. In mice, lung injury was induced with tracheal porcine pancreatic elastase. Cela1 began increasing at 21-days, and Cela1−/−mice were protected from continued airspace enlargement at 42 and 84 days (p<0.01). Aged Cela1−/−mice had less airspace simplification than aged WT mice (p<0.05). In humans and mice, CELA1 mRNA and protein were present in subsets of conducing airway epithelial and AT2 cells. COPD lungs had 3-fold more CELA1 protein than control (p<0.05). Among COPD-associated proteases, only CELA1 was positively and significantly correlated with lung elastolytic activity (p<0.001). Rabbit polyclonal and mouse monoclonal anti-CELA1 antibodies inhibited elastolytic activity of CELA1 mRNA-high but not CELA1 mRNA-low human lungs. CELA1 mRNA levels increased exponentially with age, and smoking reduced that ratio of AAT-neutralized:native CELA1 (p<0.05). CELA1 binding to lung tissue increased 6-fold with biaxial strain (p<0.05). We propose that CELA1 predisposes to progressive emphysema via (1) increased expression with age, (2) reduced AAT neutralization with smoking, and (3) increased CELA1-binding to lung matrix with strain. Anti-CELA1 therapies may represent a novel disease modifying therapy to prevent emphysema progression.ONE SENTENCE SUMMARYWe find that Chymotrypsin-like Elastase 1 (CELA1) is responsible for progressive airspace destruction in multiple mouse emphysema models, show that human lung CELA1 expression and binding to lung matrix are associated with known emphysema risk factors, and demonstrate that anti-CELA1 antibodies largely inhibit lung elastolytic activity in CELA1 mRNA-high lung specimens.

A new serine protease family with elastase activity is produced by Streptomyces bacteria

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

A210 ADLERCREUTZIA IS DEPLETED IN UC PATIENTS, EVEN BEFORE CLINICAL DIAGNOSIS, AND INVERSELY CORRELATES WITH FECAL ELASTOLYTIC ACTIVITY

Background A combination of genetics, environmental, and immune factors contribute to the development of ulcerative colitis (UC). Host proteolytic imbalance has been reported in active UC. Preliminary results from our lab suggest microbial proteolytic activity is increased before as well as after onset of UC, and transfer of this activity to mice contributes to inflammation. Aims Our aim was to correlate the elastolytic activity of fecal samples from individuals at risk for IBD, before and after onset of ulcerative colitis, with their fecal microbiota profiles Methods We first investigated proteolytic activity in fecal samples from individuals at risk to develop UC (pre-UC, n=12) prior to disease onset and after UC diagnosis (post-UC, n=7) and matched healthy controls (n=66). Microbial community analysis was performed by sequencing the V4 region of the 16S rRNA gene region using Illumina MiSeq platform. Sequences were analyzed with QIIMEv1.9.0. We measured bacterial proteolytic activity, using a FITC-elastin assay. Results Microbial community analysis revealed that the overall diversity (both richness and evenness) in UC patients was decreased compared to healthy controls as well as pre-UC patients. The relative abundance of the genus Adlercreutzia was decreased by 3.1 fold in pre-UC patients compared to healthy controls and was further decreased in post-UC (3.8 fold). The presence of Adlercreutzia was also found to be negatively correlated (r=-0.47, p&lt;0.0001) with elastolytic activity in stool supernatant, suggesting a possible protective role in the disease. Conclusions We found novel potentially protective bacteria, Adlercreutzia, which was depleted in UC patients, even before clinical diagnosis and correlated negatively with proinflammatory elastolytic activity described previously in IBD. The protective mechanisms are under investigation. On behalf of the CCC-GEM Project consortiumand Supported by a CCC GIA to EFV Funding Agencies CCC

Characterization of cathepsin S exosites that govern its elastolytic activity

We have previously determined that the elastolytic activities of cathepsins (Cat) K and V require two exosites sharing the same structural localization on both enzymes. The structural features involved in the elastolytic activity of CatS have not yet been identified. We first mutated the analogous CatK and V putative exosites of CatS into the elastolytically inactive CatL counterparts. The modification of the exosite 1 did not affect the elastase activity of CatS whilst mutation of the Y118 of exosite 2 decreased the cleavage of elastin by ∼70% without affecting the degradation of other macromolecular substrates (gelatin, thyroglobulin). T06, an ectosteric inhibitor that disrupt the elastolytic activity of CatK, blocked ∼80% of the elastolytic activity of CatS without blocking the cleavage of gelatin and thyroglobulin. Docking studies showed that T06 preferentially interacts with a binding site located on the Right domain of the enzyme, outside of the active site. The structural examination of this binding site showed that the loop spanning the L174N175G176K177 residues of CatS is considerably different from that of CatL. Mutation of this loop into the CatL-like equivalent decreased elastin degradation by ∼70% and adding the Y118 mutation brought down the loss of elastolysis to ∼80%. In addition, the Y118 mutation selectively reduced the cleavage of the basement membrane component laminin by ∼50%. In summary, our data show that the degradation of elastin by CatS requires two exosites where one of them is distinct from those of CatK and V whilst the cleavage of laminin requires only one exosite.