Quansheng Zhu

Identification of the Immunodominant Epitope Region in Phospholipase A2 Receptor-Mediating Autoantibody Binding in Idiopathic Membranous Nephropathy

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Recent clinical studies established that >70% of patients with idiopathic (also called primary) MN (IMN) possess circulating autoantibodies targeting the M-type phospholipase A2 receptor-1 (PLA2R) on the surface of glomerular visceral epithelial cells (podocytes). In situ, these autoantibodies trigger the formation of immune complexes, which are hypothesized to cause enhanced glomerular permeability to plasma proteins. Indeed, the level of autoantibody in circulation correlates with the severity of proteinuria in patients. The autoantibody only recognizes the nonreduced form of PLA2R, suggesting that disulfide bonds determine the antigenic epitope conformation. Here, we identified the immunodominant epitope region in PLA2R by probing isolated truncated PLA2R extracellular domains with sera from patients with IMN that contain anti-PLA2R autoantibodies. Patient sera specifically recognized a protein complex consisting of the cysteine-rich (CysR), fibronectin-like type II (FnII), and C-type lectin-like domain 1 (CTLD1) domains of PLA2R only under nonreducing conditions. Moreover, absence of either the CysR or CTLD1 domain prevented autoantibody recognition of the remaining domains. Additional analysis suggested that this three-domain complex contains at least one disulfide bond required for conformational configuration and autoantibody binding. Notably, the three-domain complex completely blocked the reactivity of autoantibodies from patient sera with the full-length PLA2R, and the reactivity of patient sera with the three-domain complex on immunoblots equaled the reactivity with full-length PLA2R. These results indicate that the immunodominant epitope in PLA2R is exclusively located in the CysR-FnII-CTLD1 region.

Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

NBCe1-A electrogenically cotransports Na+ and HCO3− across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA)) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser427, TM1; Thr485 and Gly486, TM3; Arg510 and Leu522, TM4; Ala799, TM10; and Arg881, TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane-processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by (2-aminoethyl)methanethiosulfonate. Functional analysis of Thr485 with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. Our findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.

NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway *

The electrogenic Na+/\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100° arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong δ- helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a δ- helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.

Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.

Structure, function, and regulation of the SLC4 NBCe1 transporter and its role in causing proximal renal tubular acidosis

Purpose of reviewThere has been significant progress in our understanding of the structural and functional properties and regulation of the electrogenic Na+-base transporter NBCe1, a membrane transporter that plays a key role in renal acid–base physiology. The NBCe1 variant NBCe1-A mediates basolateral electrogenic Na+-base transport in the proximal tubule and is critically required for transepithelial bicarbonate absorption. Mutations in NBCe1 cause autosomal recessive proximal renal tubular acidosis (pRTA). The review summarizes recent advances in this area. Recent findingsA topological model of NBCe1 has been established that provides a foundation for future structure-functional studies of the transporter. Critical residues and regions have been identified in NBCe1 that play key roles in its structure, function (substrate transport, electrogenicity) and regulation. The mechanisms of how NBCe1 mutations cause pRTA have also recently been elucidated. SummaryGiven the important role of proximal tubule transepithelial bicarbonate absorption in systemic acid–base balance, a clear understanding of the structure–functional properties of NBCe1 is a prerequisite for elucidating the mechanisms of defective transepithelial bicarbonate transport in pRTA.

Topology of NBCe1 Protein Transmembrane Segment 1 and Structural Effect of Proximal Renal Tubular Acidosis (pRTA) S427L Mutation

Background: NBCe1-A-TM1 is involved in forming part of the ion permeation pathway. Results: NBCe1-A-TM1 contains 31 amino acids. The pRTA S427L mutation alters the normal aqueous accessibility of specific TM1 residues. Conclusion: NBCe1-A-TM1 is tilted in the lipid bilayer, and its N terminus interacts with the cytoplasmic domain. The presence of S427L altered NBCe1-A-TM1 orientation. Significance: Our findings provide novel insights into the pathogenic mechanism of pRTA. In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3− from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389–Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394–Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.

Integrin αIIbβ3 inside-out activation: An in situ conformational analysis reveals a new mechanism

Background: The transmembrane domain of integrins plays a critical role in mediating receptor inside-out activation. Results: Inside-out activation triggers the repartitioning of the intracellular border of αIIb but not the β3 transmembrane domain into the lipid bilayer in living cells. Conclusion: Complex conformational changes occur in the transmembrane domain upon integrin αIIbβ3 inside-out activation. Significance: Our findings represent a new mechanism for integrin inside-out activation. Integrins are a family of heterodimeric adhesion receptors that transmit signals bi-directionally across the plasma membranes. The transmembrane domain (TM) of integrin plays a critical role in mediating transition of the receptor from the default inactive to the active state on the cell surfaces. In this study, we successfully applied the substituted cysteine scanning accessibility method to determine the intracellular border of the integrin αIIbβ3 TM in the inactive and active states in living cells. We examined the aqueous accessibility of 75 substituted cysteines comprising the C terminus of both αIIb and β3 TMs, the intracellular membrane-proximal regions, and the whole cytoplasmic tails, to the labeling of a membrane-permeable, cysteine-specific chemical biotin maleimide (BM). The active state of integrin αIIbβ3 heterodimer was generated by co-expression of activating partners with the cysteine-substituted constructs. Our data revealed that, in the inactive state, the intracellular lipid/aqueous border of αIIb TM was at Lys994 and β3 TM was at Phe727 respectively; in the active state, the border of αIIb TM shifted to Pro998, whereas the border of β3 TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon αIIbβ3 inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin αIIbβ3 and by inference, all of the integrins in their native cellular environment.

Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties.

NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties.

Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.