Wentian Luo

Kinetics in parasite abundance in susceptible and resistant mice infected with an avirulent strain of Toxoplasma gondii by using quantitative competitive PCR

The kinetics of changes in Toxoplasma gondii abundance were evaluated with a quantitative competitive (QC)-polymerase chain reaction (PCR) assay at various sites in both C57BL/6 and BALB/c mice. Higher mortality was apparent in C57BL/6 mice than in BALB/c mice when infected with a high dose of cysts. There were significant differences in cyst number when infected with a low dose of cysts, although there was no significant difference in mortality between the 2 mouse strains. One day after infection with a low dose of an avirulent Fukaya strain, T. gondii was detected in peripheral blood, mesenteric lymph nodes, spleen, lungs, and brain. Two weeks after infection, the number of T. gondii in the brain greatly increased in C57BL/6 mice but not in BALB/c mice. Thus, it would appear that the first to second week after infection is a critical period in determining T. gondii abundance. QC PCR allows the detection of low numbers of T. gondii at an early stage of infection in the murine model. This is useful for the early diagnosis of toxoplasmosis and to understand reactivation of toxoplasmosis.

Substrate specificity of inner membrane peptidase in yeast mitochondria

The inner membrane protease (IMP) cleaves intra-organelle sorting peptides from precursor proteins in mitochondria of the yeast Saccharomyces cerevisiae. An unusual feature of the IMP is the presence of two catalytic subunits, Imp1p and Imp2p, which recognize distinct substrate sets even though both enzymes belong to the same protease family. This nonoverlapping substrate specificity was hypothesized to result from the recognition of distinct residues at the P′1 position (also termed +1 position) in the protease substrates. Here, we constructed an extensive series of mutations to obtain a profile of the critical cleavage site residues in IMP substrates and conclude that Imp1p, and not Imp2p, recognizes specific P′1 residues. In addition to its specificity for P′1 residues, Imp1p also shows substrate specificity for the P3 (−3) position. In contrast, Imp2p recognizes the P1 (−1) position and the P3 position. Based on this new understanding of IMP substrate specificity, we conducted a survey for candidate IMP substrates in mammalian mitochondria and found consensus Imp2p cleavage sites in mammalian precursors to cytochrome c1 and glycerol-3-phosphate (G-3-P) dehydrogenase. Presence of a putative Imp2p cleavage site in G-3-P dehydrogenase was surprising, as its yeast ortholog contains an Imp1p cleavage site. To address this issue experimentally, we performed the first co-expression of mammalian IMP with proposed mammalian IMP precursors in yeast and show that murine precursors to cytochrome c1 and G-3-P dehydrogenase are cleaved by murine Imp2p. These results suggest, surprisingly, G-3-P dehydrogenase has switched from Imp1p in yeast to Imp2p in mammals.

Alport Alloantibodies but Not Goodpasture Autoantibodies Induce Murine Glomerulonephritis: Protection by Quinary Crosslinks Locking Cryptic α3(IV) Collagen Autoepitopes In Vivo

The noncollagenous (NC1) domains of α3α4α5(IV) collagen in the glomerular basement membrane (GBM) are targets of Goodpasture autoantibodies or Alport posttransplant nephritis alloantibodies mediating rapidly progressive glomerulonephritis. Because the autoepitopes but not the alloepitopes become cryptic upon assembly of α3α4α5NC1 hexamers, we investigated how the accessibility of B cell epitopes in vivo influences the development of glomerulonephritis in mice passively immunized with human anti-GBM Abs. Alport alloantibodies, which bound to native murine α3α4α5NC1 hexamers in vitro, deposited linearly along the mouse GBM in vivo, eliciting crescentic glomerulonephritis in Fcgr2b−/− mice susceptible to Ab-mediated inflammation. Goodpasture autoantibodies, which bound to murine α3NC1 monomer and dimer subunits but not to native α3α4α5NC1 hexamers in vitro, neither bound to the mouse GBM in vivo nor induced experimental glomerulonephritis. This was due to quinary NC1 crosslinks, recently identified as sulfilimine bonds, which comprehensively locked the cryptic Goodpasture autoepitopes in the mouse GBM. In contrast, non-crosslinked α3NC1 subunits were identified as a native target of Goodpasture autoantibodies in the GBM of squirrel monkeys, a species susceptible to Goodpasture autoantibody-mediated nephritis. Thus, crypticity of B cell autoepitopes in tissues uncouples potentially pathogenic autoantibodies from autoimmune disease. Crosslinking of α3α4α5NC1 hexamers represents a novel mechanism averting autoantibody binding and subsequent tissue injury by posttranslational modifications of an autoantigen.

Proteolysis Breaks Tolerance Toward Intact α345(IV) Collagen, Eliciting Novel Anti–Glomerular Basement Membrane Autoantibodies Specific for α345NC1 Hexamers

Goodpasture disease is an autoimmune kidney disease mediated by autoantibodies against noncollagenous domain 1 (NC1) monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis (GN). We identified a novel type of human IgG4-restricted anti–GBM autoantibodies associated with mild nonprogressive GN, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti–GBM autoantibodies were investigated in mouse models recapitulating this phenotype. Wild-type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoantibodies specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause GN. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG Abs specific for α345NC1 hexamers, which were not subclass restricted. As heterologous Ag in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a, and IgG2b autoantibodies specific for α345NC1 hexamers and induced anti–GBM Ab GN. These findings indicate that tolerance toward autologous intact α345(IV) collagen is established in hosts expressing this Ag, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α345NC1 hexamers. This provides a mechanism eliciting autoantibodies specific for α345NC1 hexamers, which are restricted to noninflammatory IgG subclasses and are nonnephritogenic. In Alport syndrome, lack of tolerance toward α345(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including proinflammatory IgG subclasses that mediate posttransplant anti–GBM nephritis.

Murine Membranous Nephropathy: Immunization with α3(IV) Collagen Fragment Induces Subepithelial Immune Complexes and FcγR-Independent Nephrotic Syndrome

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults and a significant cause of end-stage renal disease, yet current therapies are nonspecific, toxic, and often ineffective. The development of novel targeted therapies requires a detailed understanding of the pathogenic mechanisms, but progress is hampered by the lack of a robust mouse model of disease. We report that DBA/1 mice as well as congenic FcγRIII−/− and FcRγ−/− mice immunized with a fragment of α3(IV) collagen developed massive albuminuria and nephrotic syndrome, because of subepithelial deposits of mouse IgG and C3 with corresponding basement membrane reaction and podocyte foot process effacement. The clinical presentation and histopathologic findings were characteristic of MN. Although immunized mice produced genuine anti-α3NC1 autoantibodies that bound to kidney and lung basement membranes, neither crescentic glomerulonephritis nor alveolitis ensued, likely because of the predominance of mouse IgG1 over IgG2a and IgG2b autoantibodies. The ablation of activating IgG Fc receptors did not ameliorate injury, implicating subepithelial deposition of immune complexes and consequent complement activation as a major effector pathway. We have thus established an active model of murine MN. This model, leveraged by the availability of genetically engineered mice and mouse-specific reagents, will be instrumental in studying the pathogenesis of MN and evaluating the efficacy of novel experimental therapies.

Novel X-linked glomerulopathy is associated with a COL4A5 missense mutation in a non-collagenous interruption

A novel COL4A5 mutation causes rapid progression to end-stage renal disease in males, despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males have proteinuria, variable hematuria, and an early progression to end-stage renal disease. Renal biopsy findings include global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition. Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. The phenylalanine at position 222 is absolutely conserved among vertebrates. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. Immunostaining for α5(IV) collagen in renal biopsies from affected males was normal. This mutation, in a non-collagenous interruption associated with severe renal disease, provides evidence for the importance of this structural motif and suggests the range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. Hence, COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a presentation distinct from Alport syndrome.

Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

Factors Governing Nonoverlapping Substrate Specificity by Mitochondrial Inner Membrane Peptidase

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the −1 and −3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.

Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells

Abstract Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of ‘targeted-only’ cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.

Quaternary Epitopes of α345(IV) Collagen Initiate Alport Post-Transplant Anti-GBM Nephritis

Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of α5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct α345(IV) and α1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within α5NC1 monomers, shared by α345NC1 and α1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of α345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary α345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared α5NC1 alloepitopes. These results imply that quaternary epitopes within α345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, α345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas α1256NC1 hexamers become secondary targets of anti-α5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using α345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.