Repertoires of SARS-CoV-2 epitopes targeted by antibodies vary according to severity of COVID-19.

Antibodies to SARS-CoV-2 are central to recovery and immunity from COVID-19. However, the relationship between disease severity and the repertoire of antibodies against specific SARS-CoV-2 epitopes an individual develops following exposure remains incompletely understood. Here, we studied seroprevalence of antibodies to specific SARS-CoV-2 and other betacoronavirus antigens in a well-annotated, community sample of convalescent and never-infected individuals obtained in August 2020. One hundred and twenty-four participants were classified into five groups: previously exposed but without evidence of infection, having no known exposure or evidence of infection, seroconverted without symptoms, previously diagnosed with symptomatic COVID-19, and recovered after hospitalization with COVID-19. Prevalence of IgGs specific to the following antigens was compared between the five groups: recombinant SARS-CoV-2 and betacoronavirus spike and nucleocapsid protein domains, peptides from a tiled array of 22-mers corresponding to the entire spike and nucleocapsid proteins, and peptides corresponding to predicted immunogenic regions from other proteins of SARS-CoV-2. Antibody abundance generally correlated positively with severity of prior illness. A number of specific immunogenic peptides and some that may be associated with milder illness or protection from symptomatic infection were identified. No convincing association was observed between antibodies to Receptor Binding Domain(s) (RBDs) of less pathogenic betacoronaviruses HKU1 or OC43 and COVID-19 severity. However, apparent cross-reaction with SARS-CoV RBD was evident and some predominantly asymptomatic individuals had antibodies to both MERS-CoV and SARS-CoV RBDs. Findings from this pilot study may inform development of diagnostics, vaccines, and therapeutic antibodies, and provide insight into viral pathogenic mechanisms.

Paralytic shellfish toxins inhibit copper uptake in Chlamydomonas reinhardtii.

Paralytic shellfish toxins are secondary metabolites produced by several species of dinoflagellates and cyanobacteria. Known targets of these toxins, which typically occur at detrimental concentrations during harmful algal blooms, include voltage-gated ion channels in humans and other mammals. However, the effects of the toxins on the co-occurring phytoplankton community remain unknown. The present study examined the molecular mechanisms of the model photosynthetic alga Chlamydomonas reinhardtii in response to saxitoxin exposure as a means of gaining insight into the phytoplankton community response to a bloom. Previous work with yeast indicated that saxitoxin inhibited copper uptake, so experiments were designed to examine whether saxitoxin exhibited a similar mode of action in algae. Expression profiling following exposure to saxitoxin or a copper chelator produced similar profiles in copper homeostasis genes, notably induction of the cytochrome c6 (CYC6) and copper transporter (COPT1, CTR1) genes. Cytochrome c6 is used as an alternative to plastocyanin under conditions of copper deficiency, and immunofluorescence data showed this protein to be present in a significantly greater proportion of saxitoxin-exposed cells compared to controls. Live-cell imaging with a copper-sensor probe for intracellular labile Cu(I) confirmed that saxitoxin blocked copper uptake. Extrapolations of these data to phytoplankton metabolic processes along with the copper transporter as a molecular target of saxitoxin based on existing structural models are discussed.

Na,K-ATPase from mice lacking the gamma subunit (FXYD2) exhibits altered Na+ affinity and decreased thermal stability.

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth.

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V(max) conditions with little or no change in the amounts of alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.

FXYD proteins as regulators of the Na,K-ATPase in the kidney.

The FXYD gene family has seven members in mammals and others in fish. Five of these (FXYD1, FXYD2, FXYD4, FXYD7, and PLMS from shark) have been shown to alter the activity of the Na,K-ATPase, as described by other papers in this volume. The gene structure of FXYD family members suggests assembly from protein domain modules and gene duplication. The gamma subunit is unique in the family for having alternative splice variants in the coding region and can be posttranslationally modified with different final consequences for enzyme properties. The nonoverlapping distribution of gamma and CHIF (FXYD4) in kidney helps to explain physiological differences in Na(+) affinity among nephron segments. We also detected phospholemman (FXYD1) in kidney. By immunofluorescence, it was found in extraglomerular mesangial cells (EM cells) of the juxtaglomerular apparatus and in the afferent arteriole. Contrary to many reports that only alpha1 and beta1 are expressed in the kidney, we found that alpha2 and beta2 are present, although not in any nephron segment. Both were detected in arterioles, and beta2 was found in the EM cells. In contrast, alpha1, beta1, and gamma were found in adjacent macula densa. Phospholemman, alpha2, and beta2 are proposed to have distinct roles in regulating the sodium pump in structures involved in tubuloglomerular feedback.

Gamma structural variants differentially regulate Na,K-ATPase properties.

Renal Na,K-ATPase is tightly bound to a small regulatory protein, the gamma subunit (FXYD2). In rat, it occurs in two splice variants with different N-termini. Immunolocalization on kidney sections revealed distinct distribution of the gamma splice variants along the rat nephron. Where coexpressed, they coimmunoprecipitated with each other along with the alpha subunit, suggesting assembly in oligomeric complexes. Functional consequences of association with gamma were monitored in stably transfected NRK-52E cells. The outcome was that splice variants can differentially modulate the major intrinsic properties of the Na,K-ATPase under normal and stress-related conditions. The data imply an adaptive physiological mechanism of regulation of renal Na,K-ATPase through modulation of pump properties, gene expression, or both.

Phospholemman expression in extraglomerular mesangium and afferent arteriole of the juxtaglomerular apparatus.

The molecular mechanisms with which the juxtaglomerular apparatus accomplishes its twin functions, acute regulation of glomerular blood flow and secretion of renin, are still not clearly understood. Least understood is the role of the extraglomerular mesangial (EM) cells, also known as lacis or Goormaghtigh cells, which lie sandwiched between the macula densa and the afferent and efferent arterioles. Here, we report that immunoreactivity for phospholemman (FXYD1), a single-span membrane protein homologous to the gamma (gamma) sub-unit of the Na,K-ATPase, is found in the kidney in EM cells with the Na,K-ATPase beta2-subunit and in cortical blood vessels and the afferent arteriole with Na,K-ATPase alpha2 and beta2. Phospholemman's distribution in EM cells is distinct from that of the Na,K-ATPase gamma-subunit, which is found on the basolateral surface of macula densa cells with Na,K-ATPase alpha1 and beta1. Phospholemman is a major kinase target, and its location in the juxtaglomerular apparatus suggests that it is involved in tubuloglomerular feedback.

Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus.

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase gamma subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase alpha subunit isoforms (alpha1-alpha3) from cerebellum, indicating that the interaction is not specific to a particular alpha isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activity in vitro without effect on Na+ affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found.

The Na,K-ATPase alpha 2 isoform is expressed in neurons, and its absence disrupts neuronal activity in newborn mice.

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bötzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.

Distribution and oligomeric association of splice forms of Na(+)-K(+)-ATPase regulatory gamma-subunit in rat kidney.

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.