Trefoil Factor Family Domains Represent Highly Efficient Conformational Determinants for N-Linked N,N′-di-N-acetyllactosediamine (LacdiNAc) Synthesis

The disaccharide N,N′-di-N-acetyllactose diamine (LacdiNAc, GalNAcβ1–4GlcNAcβ) is found in a limited number of extracellular matrix glycoproteins and neuropeptide hormones indicating a protein-specific transfer of GalNAc by the glycosyltransferases β4GalNAc-T3/T4. Whereas previous studies have revealed evidence for peptide determinants as controlling elements in LacdiNAc biosynthesis, we report here on an entirely independent conformational control of GalNAc transfer by single TFF (Trefoil factor) domains as high stringency determinants. Human TFF2 was recombinantly expressed in HEK-293 cells as a wild type full-length probe (TFF2-Fl, containing TFF domains P1 and P2), as single P1 or P2 domain probes, as a series of Cys/Gly mutant forms with aberrant domain structures, and as a double point-mutated probe (T68Q/F59Q) lacking aromatic residues within a hydrophobic patch. The N-glycosylation probes were analyzed by mass spectrometry for their glycoprofiles. In agreement with natural gastric TFF2, the recombinant full-length and single domain probes expressed nearly exclusively fucosylated LacdiNAc on bi-antennary complex-type chains indicating that a single TFF domain was sufficient to induce transfer of this modification. Contrasting to this, the Cys/Gly mutants showed strongly reduced LacdiNAc levels and instead preponderant LacNAc expression. The probe with point mutations of two highly conserved aromatic residues in loop 3 (T68Q/F59Q) revealed that these are essential determinant components, as the probe lacked LacdiNAc expression. The structural features of the LacdiNAc-inducing determinant on human TFF2 are discussed on the basis of crystal structures of porcine TFF2, and a series of extracellular matrix-related LacdiNAc-positive glycoproteins detected as novel candidate proteins in the secretome of HEK-293 cells.     

C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6′-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.     

The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

The flagellum is a major virulence factor of motile pathogenic bacteria. This structure requires more than 50 proteins for its biogenesis and function, one of which is FlgJ. Homologs of FlgJ produced by the β- and γ-proteobacteria, such as Salmonella enterica, Vibrio spp., and both Sphingomonas sp. and Pseudomonas spp. are bifunctional, possessing an N-terminal domain responsible for proper rod assembly and a C-terminal domain possessing peptidoglycan lytic activity. Despite the amount of research conducted on FlgJ from these and other bacteria over the past 15 years, no biochemical analysis had been conducted on any FlgJ and consequently confusion exists as to whether the enzyme is a peptidoglycan hydrolase or a lytic transglycosylase. In this study, we present the development of a novel assay for glycoside lytic enzymes and its use to provide the first enzymatic characterization of the lytic domain of FlgJ from S. enterica as the model enzyme. Surprisingly, FlgJ functions as neither a muramidase nor a lytic transglycosylases but rather as a β-N-acetylglucosaminidase. As such, FlgJ represents the first autolysin with this activity to be characterized from a Gram-negative bacterium. At its optimal pH of 4.0, the Michaelis-Menten parameters of K_m and k_cat for FlgJ from S. enterica were determined to be 0.64 ± 0.18 mg ml⁻¹ and 0.13 ± 0.016 s⁻¹, respectively, using purified PG as substrate. Its catalytic residues were identified as Glu¹⁸⁴ and Glu²²³.     

云南割手密血缘F1代创新种质的因子和聚类分析

本试验对70份云南割手密血缘F1代创新种质材料8个工农艺性状进行了因子和聚类分析。因子分析中8个公因子保留前3个公因子,其累计贡献率达79.35%。第1公因子中载荷值较大的是单产、含糖量、公顷有效茎数、出苗率和分蘖率等性状;第2公因子中起主导作用的性状是茎径和株高两个产量因子;第3公因子只有11月理论蔗糖分起主导作用。以70份创新材料3个公因子的因子得分为指标,采用系统聚类中的最长距离法进行系统聚类分析。在遗传距离2.4处,参试材料被聚为十类,其中占参试材料总数的38.6%的第Ⅰ、Ⅳ、Ⅶ、Ⅷ、Ⅹ五类材料,表现高产;占参试材料82.8%的第Ⅰ、Ⅱ、Ⅳ、Ⅴ、Ⅵ、Ⅶ、Ⅸ七类材料,表现高糖,特别是其中占参试材料52.8%的Ⅰ、Ⅳ、Ⅴ、Ⅵ四类材料,11月理论蔗糖分均高于12%;占参试材料总数30%的第I、Ⅳ、Ⅶ三类材料,表现高产、高糖。这个分类结果为有针对性的利用这些创新种质材料培育高产、高糖创新亲本提供了科学依据。     

Warming temperatures and smaller body sizes: synchronous changes in growth of North Sea fishes

Decreasing body size has been proposed as a universal response to increasing temperatures. The physiology behind the response is well established for ectotherms inhabiting aquatic environments: as higher temperatures decrease the aerobic capacity, individuals with smaller body sizes have a reduced risk of oxygen deprivation. However, empirical evidence of this response at the scale of communities and ecosystems is lacking for marine fish species. Here, we show that over a 40‐year period six of eight commercial fish species in the North Sea examined underwent concomitant reductions in asymptotic body size with the synchronous component of the total variability coinciding with a 1–2 °C increase in water temperature. Smaller body sizes decreased the yield‐per‐recruit of these stocks by an average of 23%. Although it is not possible to ascribe these phenotypic changes unequivocally to temperature, four aspects support this interpretation: (i) the synchronous trend was detected across species varying in their life history and life style; (ii) the decrease coincided with the period of increasing temperature; (iii) the direction of the phenotypic change is consistent with physiological knowledge; and (iv) no cross‐species synchrony was detected in other species‐specific factors potentially impacting growth. Our findings support a recent model‐derived prediction that fish size will shrink in response to climate‐induced changes in temperature and oxygen. The smaller body sizes being projected for the future are already detectable in the North Sea.