The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph ...include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Main conclusion
Several pine members of the gymnosperm-specific GT61 clades were demonstrated to be arabinosyltransferases and xylosyltransferases catalyzing the transfer of 2-
O
-Ara
f
, 3-
O
-Ara
f
...and 2-
O
-Xyl side chains onto xylooligomer acceptors, indicating their possible involvement in Ara
f
and Xyl substitutions of xylan in pine.
Xylan in conifer wood is substituted at
O
-2 with methylglucuronic acid (MeGlcA) as well as at
O
-3 with arabinofuranose (Ara
f
), which differs from xylan in dicot wood that is typically decorated with MeGlcA but not Ara
f
. Currently, glycosyltransferases responsible for conifer xylan arabinosylation have not been identified. Here, we investigated the roles of pine glycosyltransferase family 61 (GT61) members in xylan substitutions. Biochemical characterization of four pine wood-associated GT61 members showed that they exhibited three distinct glycosyltransferase activities involved in xylan substitutions. Two of them catalyzed the addition of 2-
O
-α-Ara
f
or 3-
O
-α-Ara
f
side chains onto xylooligomer acceptors and thus were named
Pinus taeda
xylan 2-
O
-arabinosyltransferase 1 (PtX2AT1) and 3-
O
-arabinosyltransferase 1 (PtX3AT1), respectively. Two other pine GT61 members were found to be xylan 2-
O
-xylosyltransferases (PtXYXTs) adding 2-
O
-β-Xyl side chains onto xylooligomer acceptors. Furthermore, sequential reactions with PtX3AT1 and the PtGUX1 xylan glucuronyltransferase demonstrated that PtX3AT1 could efficiently arabinosylate glucuronic acid (GlcA)-substituted xylooligomers and likewise, PtGUX1 was able to add GlcA side chains onto 3-
O
-Ara
f
-substituted xylooligomers. Phylogenetic analysis revealed that PtX2AT1, PtX3AT1 and PtXYXTs resided in three gymnosperm-specific GT61 clades that are separated from the grass-expanded GT61 clade harboring xylan 3-
O
-arabinosyltransferases and 2-
O
-xylosyltransferases, suggesting that they might have been recruited independently for xylan substitutions in gymnosperms. Together, our findings have established several pine GT61 members as xylan 2-
O
- and 3-
O
-arabinosyltransferases and 2-
O
-xylosyltransferases and they indicate that pine xylan might also be substituted with 2-
O
-Ara
f
and 2-
O
-Xyl side chains.
ABSTRACT
Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG type consisting of repeating units of three consecutive xylosylated Glc ...residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1 and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1, but not AtXXT5, were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.
Abstract
Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of β-1,4-linked xylosyl residues that are often substituted with ...monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel β-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan.
Main conclusion
We have demonstrated that the Arabidopsis
FRA9
(
fragile fiber 9
) gene is specifically expressed in secondary wall-forming cells and essential for the synthesis of the unique xylan ...reducing end sequence.
Xylan is made of a linear chain of β-1,4-linked xylosyl (Xyl) residues that are often substituted with (methyl)glucuronic acid (Me)GlcA side chains and may be acetylated at
O
-2 and/or
O
-3. The reducing end of xylan from gymnosperms and dicots contains a unique tetrasaccharide sequence consisting of β-D-Xyl
p
-(1 → 3)-α-L-Rha
p
-(1 → 2)-α-D-Gal
p
A-(1 → 4)-D-Xyl
p
, the synthesis of which requires four different glycosyltransferase activities. Genetic analysis in
Arabidopsis thaliana
has so far implicated three glycosyltransferase genes,
FRA8
(
fragile fiber 8
),
IRX8
(
irregular xylem 8
) and
PARVUS
, in the synthesis of this unique xylan reducing end sequence. Here, we report the essential role of FRA9, a member of glycosyltransferase family 106 (GT106), in the synthesis of this sequence. The expression of the
FRA9
gene was shown to be induced by secondary wall master transcriptional regulators and specifically associated with secondary wall-forming cells, including xylem and fiber cells. T-DNA knockout mutation of the
FRA9
gene caused impaired secondary cell wall thickening in leaf veins and a severe arrest of plant growth. RNA interference (RNAi) downregulation of
FRA9
led to a significant reduction in secondary wall thickening of fibers, a deformation of xylem vessels and a decrease in xylan content. Structural analysis of xylanase-released xylooligomers revealed that RNAi downregulation of
FRA9
resulted in a diminishment of the unique xylan reducing end sequence and complete methylation of xylan GlcA side chains, chemotypes reminiscent of those of the
fra8
,
irx8
and
parvus
mutants. Furthermore, two FRA9 close homologs from
Populus trichocarpa
were found to be wood-associated functional orthologs of FRA9. Together, our findings uncover a member of the GT106 family as a new player involved in the synthesis of the unique reducing end sequence of xylan.
Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the ...main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.
•A Lys311-to-glutamic acid substitution in the third kringle domain of Plg is associated with HAE.•Plm-Glu311 catalyzes BK release from HK and LK independently of PKa.
Display omitted
Extracorporeal membrane oxygenation is a life-sustaining therapy for severe respiratory failure. Extracorporeal membrane oxygenation circuits require systemic anticoagulation that creates a delicate ...balance between circuit-related thrombosis and bleeding-related complications. Although unfractionated heparin is most widely used anticoagulant, alternative agents such as bivalirudin have been used. We sought to compare extracorporeal membrane oxygenation circuit thrombosis and bleeding-related outcomes in respiratory failure patients receiving either unfractionated heparin or bivalirudin for anticoagulation on venovenous extracorporeal membrane oxygenation support.
Retrospective cohort study.
Single-center, cardiothoracic ICU.
Consecutive patients requiring venovenous extracorporeal membrane oxygenation who were maintained on anticoagulation between 2013 and 2020.
IV bivalirudin or IV unfractionated heparin.
Primary outcomes were the presence of extracorporeal membrane oxygenation in-circuit-related thrombotic complications and volume of blood products administered during extracorporeal membrane oxygenation duration. One hundred sixty-two patients receiving unfractionated heparin were compared with 133 patients receiving bivalirudin for anticoagulation on venovenous extracorporeal membrane oxygenation. In patients receiving bivalirudin, there was an overall decrease in the number of extracorporeal membrane oxygenation circuit thrombotic complications (p < 0.005) and a significant increase in time to circuit thrombosis (p = 0.007). Multivariable Cox regression found that heparin was associated with a significant increase in risk of clots (ExpB = 2.31, p = 0.001). Patients who received bivalirudin received significantly less volume of packed RBCs, fresh frozen plasma, and platelet transfusion (p < 0.001 for each). There was a significant decrease in the number major bleeding events in patients receiving bivalirudin, 40.7% versus 11.7%, p < 0.001.
Patients receiving bivalirudin for systemic anticoagulation on venovenous extracorporeal membrane oxygenation experienced a decrease in the number of extracorporeal membrane oxygenation circuit-related thrombotic events as well as a significant decrease in volume of blood products administered.
Abstract Grass xylan consists of a linear chain of β-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or glucuronic acid (GlcA)/methylglucuronic ...acid (MeGlcA) residues, and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remains elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto (Araf)-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution patterns of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.
•Encapsulated blueberry extract had higher antioxidant capacity than pomace extract.•Encapsulated blueberry extract had comparable properties to freeze-dried juice.•Anthocyanidin-like compounds may ...form during digestion of blueberry extracts.
We aimed to determine the effect of encapsulation on the release properties of blueberry extracts during simulated gastrointestinal digestion. An ethanolic pomace extract was microencapsulated with whey protein isolate via spray drying. The in vitro release of monomeric anthocyanins, phenolics and ferric reducing antioxidant activity of the microcapsules (W) were evaluated for the microcapsules and two non-encapsulated systems: ethanolic pomace extract (P) and freeze-dried juice (F). Concentrations of anthocyanin and phenolics were normalised prior to digestion. Results showed that antioxidant activity was in the order of: F>W>P. Regardless of encapsulation, more phenolics were released from W and P than F. Anthocyanin concentration decreased after intestinal digestion for W, but remained constant for P and F. MALDI-MS showed similar spectra for P and F but not for W. The spray-dried product has comparable release characteristics to freeze-dried juice, and may be investigated for food applications.
Main conclusion
A member of the rice GT61 clade B is capable of transferring both 2-
O
-xylosyl and 2-
O
-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-
O
...-xylosyl residue onto xylan.
Grass xylan is substituted predominantly with 3-
O
-arabinofuranose (Ara
f
) as well as with some minor side chains, such as 2-
O
-Ara
f
and 2-
O
-(methyl)glucuronic acid (Me)GlcA. 3-
O
-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-
O
-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Ara
f
residues from UDP-Xyl and UDP-Ara
f
, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Ara
f
residues were transferred onto
O
-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-
O
-xylosyl/2-
O
-arabinosyl transferase and OsXYXT2 is a xylan 2-
O
-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.
Graphical Abstract
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