Search for associations between polymorphic variants of human acid chitinase gene and bronchial asthma in children of Novosibirsk

High prevalence of bronchial asthma among the population (about 300 million people all over the world) provides rationale for the search for candidate genes of disease. Human acidic chitinase (CHIA (AMCase)), encoded by the CHIA gene, is involved in the degradation of chitin, a component of the fungal cell wall and arthropod exoskeleton, which, if present in food or house dust, is a provoking factor for the bronchial asthma (BA) development. Functionally significant mutations in the CHIA gene may apparently increase the risk of susceptibility to BA.Aim. The aim of the study was to assess the associations of single nucleotide polymorphisms (SNPs) rs12033184 and rs3806448 in the CHIA gene with bronchial asthma in children in Novosibirsk.Material and Methods. The study was organized as case-control. A total of 537 blood samples were used. SNPs were determined by real-time PCR. The associations of polymorphic variants with the disease were assessed by the odds ratio.Results. No associations of rs12033184 and rs3806448 with BA were found.Conclusion. The role of acidic chitinase gene in the development of BA in residents of Novosibirsk was found to be less significant than in the Indian population where it was previously shown to be associated with the disease.

The ACID PHOSPHATASE 1 regulates Pi stress adaptation by maintaining intracellular Pi homeostasis

The concentration and homeostasis of intracellular phosphate (Pi) are crucial for sustaining cell metabolism and growth. During short-term Pi starvation, intracellular Pi is maintained relatively constant at the expense of vacuolar Pi. After the vacuolar stored Pi is exhausted, the plant cells induce the synthesis of intracellular acid phosphatase (APase) to recycle Pi from expendable organic phosphate (Po). In this study, the expression, enzymatic activity and subcellular localization of ACID PHOSPHATASE 1 (OsACP1) were determined. OsACP1 expression is specifically induced in almost all cell types of leaves and roots under Pi stress conditions. OsACP1 encodes an acid phosphatase with broad Po substrates and localizes in the endoplasmic reticulum (ER) and Golgi apparatus (GA). Phylogenic analysis demonstrates that OsACP1 has a similar structure with human acid phosphatase PHOSPHO1. Overexpression or mutation of OsACP1 affected Po degradation and utilization, which further influenced plant growth and productivity under both Pi-sufficient and Pi-deficient conditions. Moreover, overexpression of OsACP1 significantly affected intracellular Pi homeostasis and Pi starvation signalling. We concluded that OsACP1 is an active acid phosphatase that regulates rice growth under Pi stress conditions by recycling Pi from Po in the ER and GA.

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

Production of Human Acid-Alpha Glucosidase With a Paucimannose Structure by Glycoengineered Arabidopsis Cell Culture

Plant cell cultures have emerged as a promising platform for the production of biopharmaceutics due to their cost-effectiveness, safety, ability to control the cultivation, and secrete products into culture medium. However, the use of this platform is hindered by the generation of plant-specific N-glycans, the inability to produce essential N-glycans for cellular delivery of biopharmaceutics, and low productivity. In this study, an alternative acid-alpha glucosidase (GAA) for enzyme replacement therapy of Pompe disease was produced in a glycoengineered Arabidopsis alg3 cell culture. The N-glycan composition of the GAA consisted of a predominantly paucimannosidic structure, Man3GlcNAc2 (M3), without the plant-specific N-glycans. Supplementing the culture medium with NaCl to a final concentration of 50 mM successfully increased GAA production by 3.8-fold. GAA from an NaCl-supplemented culture showed a similar N-glycan profile, indicating that the NaCl supplementation did not affect N-glycosylation. The results of this study highlight the feasibility of using a glycoengineered plant cell culture to produce recombinant proteins for which M3 or mannose receptor-mediated delivery is desired.

Mambalgin-3 potentiates human acid-sensing ion channel 1b under mild to moderate acidosis: Implications as an analgesic lead

Acid-sensing ion channels (ASICs) are expressed in the nervous system, activated by acidosis, and implicated in pain pathways. Mambalgins are peptide inhibitors of ASIC1 and analgesic in rodents via inhibition of centrally expressed ASIC1a and peripheral ASIC1b. This activity has generated interest in mambalgins as potential therapeutics. However, most mechanism and structure–activity relationship work on mambalgins has focused on ASIC1a, and neglected the peripheral analgesic target ASIC1b. Here, we compare mambalgin potency and mechanism of action at heterologously expressed rat and human ASIC1 variants. Unlike the nanomolar inhibition at ASIC1a and rodent ASIC1b, we find mambalgin-3 only weakly inhibits human ASIC1b and ASIC1b/3 under severe acidosis, but potentiates currents under mild/moderate acidosis. Our data highlight the importance of understanding the activity of potential ASIC-targeting pharmaceuticals at human channels.

Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5′-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

High-throughput characterization of 309 photocrosslinker-bearing ASIC1a variants maps residues critical for channel function and pharmacology

Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Overall, this protocol will enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.