Association of mannose-binding lectin gene polymorphism but not of mannose-binding serine protease 2 with chronic severe aortic regurgitation of rheumatic etiology

N-Acetylglucosamine (GlcNAc) is the major immunoepitope of group A streptococcal cell wall carbohydrates. Antistreptococcal antibodies cross-reactive with anti-GlcNAc and laminin are present in sera of patients with rheumatic fever. The cross-reactivity of these antibodies with human heart valvular endothelium and the underlying basement membrane has been suggested to be a possible cause of immune-mediated valve lesion. Mannose-binding lectin (MBL) encoded by the MBL2 gene, a soluble pathogen recognition receptor, has high affinity for GlcNAc. We postulated that mutations in exon 1 of the MBL2 gene associated with a deficient serum level of MBL may contribute to chronic severe aortic regurgitation (AR) of rheumatic etiology. We studied 90 patients with severe chronic AR of rheumatic etiology and 281 healthy controls (HC) for the variants of the MBL2 gene at codons 52, 54, and 57 by using a PCR-restriction fragment length polymorphism-based method. We observed a significant difference in the prevalence of defective MBL2 alleles between patients with chronic severe AR and HC. Sixteen percent of patients with chronic severe AR were homozygotes or compound heterozygotes for defective MBL alleles in contrast to 5% for HC (P = 0.0022; odds ratio, 3.5 [ 95% confidence interval, 1.6 to 7.7]). No association was detected with the variant of the MASP2 gene. Our study suggests that MBL deficiency may contribute to the development of chronic severe AR of rheumatic etiology.

Mannose-binding lectin and MBL-associated serine protease-2 gene polymorphisms in a Brazilian population from Rio de Janeiro

Mannose-binding lectin (MBL) is a protein able to bind to carbohydrate patterns on pathogen membranes; upon MBL binding, its associated serine protease MBL-associated serine protease type 2 (MASP2) is autoactivated, promoting the activation of complement via the lectin pathway. For both MBL2 and MASP2 genes, the frequencies of polymorphisms are extremely variable between different ethnicities, and this aspect has to be carefully considered when performing genetic studies. While polymorphisms in the MBL-encoding gene (MBL2) have been associated, depending upon ethnicity, with several diseases in different populations, little is known about the distribution of MASP2 gene polymorphisms in human populations. The aim of our study was thus to determine the frequencies of MBL2 (exon 1 and promoter) and MASP2 (p.D371Y) polymorphisms in a Brazilian population from Rio de Janeiro. A total of 294 blood donor samples were genotyped for 27 polymorphisms in the MBL2 gene by direct sequencing of a region spanning from the promoter polymorphism H/L rs11003125 to the rs1800451 polymorphism (at codon 57 in the first exon of the gene). Genotyping for MASP2 p.D371Y was carried out using fluorogenic probes. To our knowledge, this is the first study reporting the prevalence of the MASP2 p.D371Y polymorphism in a Brazilian population. The C allele frequency 39% is something intermediate between the reported 14% in Europeans and 90% in Sub-Saharan Africans. MBL2 polymorphisms frequencies were quite comparable to those previously reported for admixed Brazilians. Both MBL2 and MASP2 polymorphisms frequencies reported in our study for the admixed Brazilian population are somehow intermediate between those reported in Europeans and Africans, reflecting the ethnic composition of the southern Brazilian population, estimated to derive from an admixture of Caucasian (31%), African (34%) and Native American (33%) populations. In conclusion, our population genetic study describes the frequencies of MBL2 and MASP2 functional SNPs in a population from Rio de Janeiro, with the aim of adding new information concerning the distribution of these SNPs in a previously unanalysed Brazilian population, thus providing a new genetic tool for the evaluation of the association of MBL2 and MASP2 functional SNPs with diseases in Brazil, with particular emphasis on the state of Rio de Janeiro.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Sequential origin in the high performance properties of spider dragline silk

Major ampullate (MA) dragline silk supports spider orb webs, combining strength and extensibility in the toughest biomaterial. MA silk evolved ~376 MYA and identifying how evolutionary changes in proteins influenced silk mechanics is crucial for biomimetics, but is hindered by high spinning plasticity. We use supercontraction to remove that variation and characterize MA silk across the spider phylogeny. We show that mechanical performance is conserved within, but divergent among, major lineages, evolving in correlation with discrete changes in proteins. Early MA silk tensile strength improved rapidly with the origin of GGX amino acid motifs and increased repetitiveness. Tensile strength then maximized in basal entelegyne spiders, ~230 MYA. Toughness subsequently improved through increased extensibility within orb spiders, coupled with the origin of a novel protein (MaSp2). Key changes in MA silk proteins therefore correlate with the sequential evolution high performance orb spider silk and could aid design of biomimetic fibers.