Secretory antibody formation: conserved binding interactions between J chain and polymeric Ig receptor from humans and amphibians

Abs of the secretory Ig (SIg) system reinforce numerous innate defense mechanisms to protect the mucosal surfaces against microbial penetration. SIgs are generated by a unique cooperation between two distinct cell types: plasma cells that produce polymers of IgA or IgM (collectively called pIgs) and polymeric Ig receptor (pIgR)-expressing secretory epithelial cells that mediate export of the pIgs to the lumen. Apical delivery of SIgs occurs by cleavage of the pIgR to release its extracellular part as a pIg-bound secretory component, whereas free secretory components are derived from an unoccupied receptor. The joining chain (J chain) is crucial in pIg/SIg formation because it serves to polymerize Igs and endows them with a binding site for the pIgR. In this study, we show that the J chain from divergent tetrapods including mammals, birds, and amphibians efficiently induced polymerization of human IgA, whereas the J chain from nurse shark (a lower vertebrate) did not. Correctly assembled polymers showed high affinity to human pIgR. Sequence analysis of the J chain identified two regions, conserved only in tetrapods, which by mutational analysis were found essential for pIgA-pIgR complexing. Furthermore, we isolated and characterized pIgR from the amphibian Xenopus laevis and demonstrated that its pIg binding domain showed high affinity to human pIgA. These results showed that the functional site of interaction between pIgR, J chain and Ig H chains is conserved in these species and suggests that SIgs originated in an ancestor common to tetrapods.     

Vaccinia virus-specific CD4+ T cell responses target a set of antigens largely distinct from those targeted by CD8+ T cell responses

Recent studies have defined vaccinia virus (VACV)-specific CD8(+) T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4(+) T cell responses. In this study, we identified 14 I-A(b)-restricted VACV-specific CD4(+) T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for approximately 20% of the total anti-VACV CD4(+) T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4(+) T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4(+) T cell targets. These results are in contrast to what was previously found in CD8(+) T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4(+) and CD8(+) T cell responses to a complex pathogen.     

Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Twospotted spider mite population level, distribution, and damage on ivy geranium in response to different nitrogen and phosphorus fertilization regimes

The influence of plant nutrition on arthropod pests has often been studied by comparing plants provided suboptimal nutrients with those provided sufficient or luxurious nutrients, but such results have limited applicability to commercially produced crops because nitrogen (N) and phosphorus (P) are almost never limiting in greenhouse production. We conducted a series of experiments with ivy geranium, Pelargonium peltatum (L.) L'H?. ex Aiton 'Amethyst 96' to determine the response of twospotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae), to six combinations of N (8 or 24 mM) and P (0.32, 0.64, or 1.28 mM) that reflected commercial production practices. All six combinations resulted in saleable plants when plants were free of spider mites, but tissue N and P concentrations among fertilizer combinations were different. On mite-infested plants, no difference in mite numbers or plant damage was found in response to N fertilization rates. Phosphorus had no effect on mite population level until week 8, at which time plants fertilized with 0.64 mM P had slightly more mites than plants fertilized with 0.32 mM. However, overall quality and dry weight of plants fertilized by 0.32 mM P was lower than that of 0.64 and 1.28 mM, which suggests that ivy geranium plants fertilized with the higher P rates may better compensate for mite feeding damage. Positive correlations were found between within-plant distribution of mites and the corresponding tissue N and P concentrations in three foliage strata, suggesting that tissue nutrient content may influence mite selection of feeding sites.     

Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway

We present the first two identified cases of phosphoserine aminotransferase deficiency. This disorder of serine biosynthesis has been identified in two siblings who showed low concentrations of serine and glycine in plasma and cerebrospinal fluid. Clinically, the index patient presented with intractable seizures, acquired microcephaly, hypertonia, and psychomotor retardation and died at age 7 mo despite supplementation with serine (500 mg/kg/d) and glycine (200 mg/kg/d) from age 11 wk. The younger sibling received treatment from birth, which led to a normal outcome at age 3 years. Measurement of phosphoserine aminotransferase activity in cultured fibroblasts in the index patient was inconclusive, but mutational analysis revealed compound heterozygosity for two mutations in the PSAT1 gene--one frameshift mutation (c.delG107) and one missense mutation (c.299A-->C [p.Asp100Ala])--in both siblings. Expression studies of the p.Asp100Ala mutant protein revealed a V(max) of only 15% of that of the wild-type protein.     

The NECAP PHear domain increases clathrin accessory protein binding potential

AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 alpha-ear, is the recruitment of accessory proteins. NECAPs are alpha-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 alpha-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the alpha-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.