Molecular Identification of Anion Exchange Protein 3 in Pacific White Shrimp (Litopenaeus vannamei): mRNA Profiles for Tissues, Ontogeny, Molting, and Ovarian Development and Its Potential Role in Stress-Induced Gill Damage

Bicarbonate (HCO3–) transport mechanisms play an essential role in the acid-base homeostasis of aquatic animals, and anion exchange protein 3 (AE3) is a membrane transport protein that exchanges Cl–/HCO3– across the cell membrane to regulate the intracellular pH. In this study, the full-length cDNA of AE3 (Lv-AE3) was obtained from the Pacific white shrimp (Litopenaeus vannamei). The Lv-AE3 cDNA is 4,943 bp in length, contains an open reading frame of 2,850 bp, coding for a protein of 949 amino acids with 12 transmembrane domains. Lv-AE3 shows high sequence homology with other AE3 at the protein level. Lv-AE3 mRNA was ubiquitously detected in all tissues selected, with the highest expression level in the gill, followed by the ovary, eyestalk and brain. By in situ hybridization, Lv-AE3-positive cells were shown predominant localization in the secondary gill filaments. The expression levels of Lv-AE3 were further investigated during the essential life processes of shrimp, including ontogeny, molting, and ovarian development. In this case, the spatiotemporal expression profiles of Lv-AE3 in L. vannamei were highly correlated with the activities of water and ion absorption; for example, increased mRNA levels were present after hatching, during embryonic development, after ecdysis during the molt cycle, and in the stage IV ovary during gonadal development. After low/high pH and low/high salinity challenges, the transcript levels of Lv-AE3 were reduced in the gill, while the cell apoptosis rate increased. In addition, knockdown of Lv-AE3 mRNA expression induced cell apoptosis in the gill, indicating a potential link between Lv-AE3 and gill damage. Altogether, this study thoroughly investigated the relationship between the mRNA expression profiles of Lv-AE3 and multiple developmental and physiological processes in L. vannamei, and it may benefit the protection of crustaceans from fluctuated aquatic environments.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton

Theileria equi (T. equi) is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

External α-carbonic anhydrase and solute carrier 4 are required for bicarbonate uptake in a freshwater angiosperm

The freshwater monocot Ottelia alismoides is the only known species to operate three CO2-concentrating mechanisms (CCMs): constitutive bicarbonate (HCO3–) use, C4 photosynthesis, and facultative Crassulacean acid metabolism, but the mechanism of HCO3– use is unknown. We found that the inhibitor of an anion exchange protein, 4,4'-diisothio-cyanatostilbene-2,2'-disulfonate (DIDS), prevented HCO3– use but also had a small effect on CO2 uptake. An inhibitor of external carbonic anhydrase (CA), acetazolamide (AZ), reduced the affinity for CO2 uptake but also prevented HCO3– use via an effect on the anion exchange protein. Analysis of mRNA transcripts identified a homologue of solute carrier 4 (SLC4) responsible for HCO3– transport, likely to be the target of DIDS, and a periplasmic α-carbonic anhydrase 1 (α-CA1). A model to quantify the contribution of the three different pathways involved in inorganic carbon uptake showed that passive CO2 diffusion dominates inorganic carbon uptake at high CO2 concentrations. However, as CO2 concentrations fall, two other pathways become predominant: conversion of HCO3– to CO2 at the plasmalemma by α-CA1 and transport of HCO3– across the plasmalemma by SLC4. These mechanisms allow access to a much larger proportion of the inorganic carbon pool and continued photosynthesis during periods of strong carbon depletion in productive ecosystems.

Transport of H2S and HS− across the human red blood cell membrane: rapid H2S diffusion and AE1-mediated Cl−/HS− exchange

The rates of H2S and HS− transport across the human erythrocyte membrane were estimated by measuring rates of dissipation of pH gradients in media containing 250 μM H2S/HS−. Net acid efflux is caused by H2S/HS− acting analogously to CO2/HCO3− in the Jacobs-Stewart cycle. The steps are as follows: 1) H2S efflux through the lipid bilayer and/or a gas channel, 2) extracellular H2S deprotonation, 3) HS− influx in exchange for Cl−, catalyzed by the anion exchange protein AE1, and 4) intracellular HS− protonation. Net acid transport by the Cl−/HS−/H2S cycle is more efficient than by the Cl−/HCO3−/CO2 cycle because of the rapid H2S-HS− interconversion in cells and medium. The rates of acid transport were analyzed by solving the mass flow equations for the cycle to produce estimates of the HS− and H2S transport rates. The data indicate that HS− is a very good substrate for AE1; the Cl−/HS− exchange rate is about one-third as rapid as Cl−/HCO3− exchange. The H2S permeability coefficient must also be high (>10−2 cm/s, half time <0.003 s) to account for the pH equilibration data. The results imply that H2S and HS− enter erythrocytes very rapidly in the microcirculation of H2S-producing tissues, thereby acting as a sink for H2S and lowering the local extracellular concentration, and the fact that HS− is a substrate for a Cl−/HCO3− exchanger indicates that some effects of exogenous H2S/HS− may not result from a regulatory role of H2S but, rather, from net acid flux by H2S and HS− transport in a Jacobs-Stewart cycle.