RESUMO
Soluble human lectins are critical components of innate immunity. Genetic models suggest that lectins influence host-resident microbiota, but their specificity for commensal and mutualist species is understudied. Elucidating lectins' roles in regulating microbiota requires an understanding of which microbial species they bind within native communities. To profile human lectin recognition, we developed Lectin-Seq. We apply Lectin-Seq to human fecal microbiota using the soluble mannose-binding lectin (MBL) and intelectin-1 (hItln1). Although each lectin binds a substantial percentage of the samples (10 to 20%), the microbial interactomes of MBL and hItln1 differ markedly in composition and diversity. MBL binding is highly selective for a small subset of species commonly associated with humans. In contrast, hItln1's interaction profile encompasses a broad range of lower-abundance species. Our data uncover stark differences in the commensal recognition properties of human lectins.
Assuntos
Imunidade Inata , Lectinas , Humanos , Lectinas/genéticaRESUMO
The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.
Assuntos
Interações entre Hospedeiro e Microrganismos , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas , Humanos , Parede Celular/metabolismo , Lectinas/metabolismo , Mucinas/metabolismo , Polissacarídeos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
Recognition of distinct glycans is central to biology, and lectins mediate this function. Lectin glycan preferences are usually centered on specific monosaccharides. In contrast, human intelectin-1 (hItln-1, also known as Omentin-1) is a soluble lectin that binds a range of microbial sugars, including ß-d-galactofuranose (ß-Galf), d-glycerol 1-phosphate, d-glycero-d-talo-oct-2-ulosonic acid (KO), and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). Though these saccharides differ dramatically in structure, they share a common feature-an exocyclic vicinal diol. How and whether such a small fragment is sufficient for recognition was unclear. We tested several glycans with this epitope and found that l-glycero-α-d-manno-heptose and d-glycero-α-d-manno-heptose possess the critical diol motif yet bind weakly. To better understand hItln-1 recognition, we determined the structure of the hItln-1·KO complex using X-ray crystallography, and our 1.59 Å resolution structure enabled unambiguous assignment of the bound KO conformation. This carbohydrate conformation was present in >97% of the KDO/KO structures in the Protein Data Bank. Bioinformatic analysis revealed that KO and KDO adopt a common conformation, while heptoses prefer different conformers. The preferred conformers of KO and KDO favor hItln-1 engagement, but those of the heptoses do not. Natural bond orbital (NBO) calculations suggest these observed conformations, including the side chain orientations, are stabilized by not only steric but also stereoelectronic effects. Thus, our data highlight a role for stereoelectronic effects in dictating the specificity of glycan recognition by proteins. Finally, our finding that hItln-1 avoids binding prevalent glycans with a terminal 1,2-diol (e.g., N-acetyl-neuraminic acid and l-glycero-α-d-manno-heptose) suggests the lectin has evolved to recognize distinct bacterial species.
Assuntos
Polissacarídeos/química , Configuração de Carboidratos , Cristalografia por Raios X , Ligação Proteica , EstereoisomerismoRESUMO
PurposeNext-generation sequencing (NGS) often identifies multiple rare predicted-deleterious variants (RDVs) in different genes associated with a recessive disorder in a given patient. Such variants have been proposed to contribute to digenicity/oligogenicity or "triallelism" or to act as genetic modifiers.MethodsUsing the recessive ciliopathy Joubert syndrome (JBTS) as a model, we investigated these possibilities systematically, relying on NGS of known JBTS genes in a large JBTS and two control cohorts.Results65% of affected individuals had a recessive genetic cause, while 4.9% were candidates for di-/oligogenicity, harboring heterozygous RDVs in two or more genes, compared with 4.2-8% in controls (P = 0.66-0.21). Based on Exome Aggregation Consortium (ExAC) allele frequencies, the probability of cumulating RDVs in any two JBTS genes is 9.3%. We found no support for triallelism, as no unaffected siblings carried the same biallelic RDVs as their affected relative. Sixty percent of individuals sharing identical causal RDVs displayed phenotypic discordance. Although 38% of affected individuals harbored RDVs in addition to the causal mutations, their presence did not correlate with phenotypic severity.ConclusionOur data offer little support for triallelism or digenicity/oligogenicity as clinically relevant inheritance modes in JBTS. While phenotypic discordance supports the existence of genetic modifiers, identifying clinically relevant modifiers remains challenging.
Assuntos
Genes Recessivos , Estudos de Associação Genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Variação Genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Cerebelo/anormalidades , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Genes Modificadores , Estudos de Associação Genética/métodos , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Modelos Genéticos , Herança Multifatorial , Mutação , Fenótipo , Retina/anormalidadesRESUMO
BACKGROUND: Joubert syndrome (JS) is a recessive ciliopathy characterised by a distinctive brain malformation 'the molar tooth sign'. Mutations in >27 genes cause JS, and mutations in 12 of these genes also cause Meckel-Gruber syndrome (MKS). The goals of this work are to describe the clinical features of MKS1-related JS and determine whether disease causing MKS1 mutations affect cellular phenotypes such as cilium number, length and protein content as potential mechanisms underlying JS. METHODS: We measured cilium number, length and protein content (ARL13B and INPP5E) by immunofluorescence in fibroblasts from individuals with MKS1-related JS and in a three-dimensional (3D) spheroid rescue assay to test the effects of disease-related MKS1 mutations. RESULTS: We report MKS1 mutations (eight of them previously unreported) in nine individuals with JS. A minority of the individuals with MKS1-related JS have MKS features. In contrast to the truncating mutations associated with MKS, all of the individuals with MKS1-related JS carry ≥ 1 non-truncating mutation. Fibroblasts from individuals with MKS1-related JS make normal or fewer cilia than control fibroblasts, their cilia are more variable in length than controls, and show decreased ciliary ARL13B and INPP5E. Additionally, MKS1 mutant alleles have similar effects in 3D spheroids. CONCLUSIONS: MKS1 functions in the transition zone at the base of the cilium to regulate ciliary INPP5E content, through an ARL13B-dependent mechanism. Mutations in INPP5E also cause JS, so our findings in patient fibroblasts support the notion that loss of INPP5E function, due to either mutation or mislocalisation, is a key mechanism underlying JS, downstream of MKS1 and ARL13B.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Cerebelo/anormalidades , Cílios/genética , Cílios/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Retina/anormalidades , Fatores de Ribosilação do ADP/metabolismo , Anormalidades Múltiplas/diagnóstico , Animais , Encéfalo/patologia , Células Cultivadas , Cerebelo/metabolismo , Cílios/patologia , Éxons , Anormalidades do Olho/diagnóstico , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Doenças Renais Císticas/diagnóstico , Imageamento por Ressonância Magnética , Camundongos , Modelos Biológicos , Mutação , Ligação Proteica , Transporte Proteico , Retina/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain â¼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.