Hydroxyproline Residues Research Articles

Toxin Homology Domain in Plant Type 2 Prolyl 4-Hydroxylases Acts as a Golgi Localization Domain.

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination.

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.

Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942cm-1 and a shoulder at 2917cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Molecular characterisation, gelation kinetics and rheological enhancement of ultrasonically extracted triggerfish skin gelatine

Improper fish skin disposal raises environmental and health concerns, highlighting the need for sustainable waste management. Turning discarded fish skins into marketable products like gelatine benefits both the economy and ecology. This study aims to investigate the feasibility of employing ultrasonic technique for the recovery of gelatine from the collagen-rich titan triggerfish skin following a preliminary pretreatment. The applied ultrasonic parameters included 100 W power, 40 kHz frequency, and a temperature held at 50 °C over a 3 h duration, resulting in a maximum gelatine yield of 20.5 ± 0.3 % (w/w) based on wet weight measurements. The biochemical and functional attributes of the extracted triggerfish gelatine (TFG) were compared to those of commercial bovine gelatine (CBG). TFG exhibited a substantial protein content (92.2 ± 0.2 %), along with low levels of moisture (7.4 ± 0.1 %), fat (0.2 ± 0.1 %), and ash (0.2 ± 0.04 %). SDS-PAGE showed that the primary elements of TFG were β- and α-chains (MW ∼ 150 to 220 kDa). While both gelatine types displayed predominant FTIR bands in the amide area (amide-I, amide-II and amide-ІІІ), TFG spectra, however, suggested that the ultrasonic treatment had no discernible effect on its triple helical structure. Significant variations in the gross amino acid profiles were found in both TFG and CBG. Nevertheless, compared to CBG, TFG had lower levels of proline and hydroxyproline residues. Additionally, TFG exhibited relatively lower gel strength; however, the optimal inclusion of microbial transglutaminase (MTGase) increased its gel strength (394±4 g) and melting temperature (Tmp = 31.9 °C), while reducing gelation time (<50 min at 20 °C). Functionally, TFG demonstrated higher foaming but marginally lower emulsifying properties than CBG. The finding underscores the potential of ultrasonication to mitigate fish skin waste emissions and efficiently retrieve high-quality gelatine within a shorter time frame.

Techno-functional characterization of gelatin extracted from the smooth-hound shark skins: Impact of pretreatments and drying methods

Gelatin derived from marine by-products could be an interesting alternative to classic mammalian gelatin. The pretreatment and extraction conditions could influence the size of the resulting peptide chains and therefore their techno-functional properties. Thus, it is important to optimize the production process to get a gelatin for the appropriate applications. Skin pretreatment was done by microwaves or oven-drying and the extracted gelatin was dried by spray- or freeze-drying. Freeze-dried gelatin extracted from untreated skin (FGUS) had the highest gelatin yield (10.40%). Gelatin proximate composition showed that proteins were the major component (87.12–89.95%), while lipids showed the lowest contents (0.65–2.26%). Glycine showed the highest level (299–316/1000 residues) in the extracted gelatins. Proline and hydroxyproline residues of gelatins from untreated skin were significantly higher than those from pretreated skin-gelatin. FTIR spectra were characterized by peaks of the amide A (3430-3284 cm−1), B (3000-2931 cm−1), I (1636–1672 cm−1), II (1539–1586 cm−1) and III (1000–1107 cm−1). Spray-drying decreased the gelling properties of gelatins, since it reduced gelling and melting temperatures compared to freeze-drying. Skin pretreatment significantly reduced the gel strength of gelatin by about 50–100 g depending on the gelatin drying method. The FGUS showed better surface properties compared to other gelatins. The highest emulsion activity index (39.42 ± 1.02 m2/g) and foaming expansion (172.33 ± 2.35%) were measured at 3% FGUS. Therefore, the promising properties of freeze-dried gelatin derived from untreated skin, gave it the opportunity to be successfully used as a techno-functional ingredient in many formulations.

Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA.

The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNaV1.4 with a 939 nM IC50. Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity.

Protein O-glycosylation regulates diverse developmental and defense processes in plants.

Post-translational modifications affect protein functions and play key roles in controlling biological processes. Plants have unique types of O-glycosylation that are different from those of animals and prokaryotes, and they play roles in modulating the functions of secretory proteins and nucleocytoplasmic proteins by regulating transcription and mediating localization and degradation. O-glycosylation is complex because of the dozens of different O-glycan types, the widespread existence of hydroxyproline (Hyp), serine (Ser), and threonine (Thr) residues in proteins attached by O-glycans, and the variable modes of linkages connecting the sugars. O-glycosylation specifically affects development and environmental acclimatization by affecting diverse physiological processes. This review describes recent studies on the detection and functioning of protein O-glycosylation in plants, and provides a framework for the O-glycosylation network that underlies plant development and resistance.

Bifidobacterial GH146 β-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-β-l-Araf-(1→2)-β-l-Araf-(1→2)-β-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 β-l-arabinobiosidase (HypBA2), producing β-l-Araf-(1→2)-l-Ara (β-l-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

First Anti-Inflammatory Peptide AnmTX Sco 9a-1 from the Swimming Sea Anemone Stomphia coccinea.

A novel peptide AnmTX Sco 9a-1 with the β-hairpin fold was isolated from the swimming sea anemone Stomphia coccinea (Actinostolidae family). The peptide consists of 28 amino acid residues, including modified hydroxyproline residue, and its measured molecular mass is 2960 Da. The peptide was not toxic on mice; however, it stimulated their exploratory motivation and active search behavior, and demonstrated an anti-anxiety effect. AnmTX Sco 9a-1 at doses of 0.1 and 1 mg/kg reduced the volume of edema during 24 h better than the nonsteroidal anti-inflammatory drug, Diclofenac, at dose of 1 mg/kg in a model of acute local λ-carrageenan-induced inflammation. ELISA analysis of the animal's blood showed that peptide at a dose of 1 mg/kg reduced the content of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator responsible in the edema development, up to the level of TNF-α in the intact group. Besides, AnmTX Sco 9a-1 demonstrated a significant analgesic effect on acute pain sensitivity in the carrageenan-induced thermal hyperalgesia model at doses of 0.1 and 1 mg/kg. Activity of AnmTX Sco 9a-1 was shown not to be associated with modulation of nociceptive ASIC channels.

Effects of Spray Drying and Freeze Drying on Physicochemical Properties, Antioxidant and ACE Inhibitory Activities of Bighead Carp (Aristichthys nobilis) Skin Hydrolysates.

The physicochemical, structural properties, antioxidant, and angiotensin I-converting enzyme (ACE) inhibitory activities of fish skin protein hydrolysate (SPH) that were freeze-dried (SPH-FD) and spray-dried (SPH-SD) were investigated. SPH-SD showed abundant volatile compounds, higher DPPH radical scavenging activity and ferrous iron chelating activity than SPH-FD, while the ABTS radical scavenging activity and ACE inhibitory activity were not influenced by the drying method. Amino acid compositions showed a higher proportion of proline and hydroxyproline residues in SPH-FD. The major molecular weights were both distributed below 1000 Da. SPH-SD had spherical structures, while SPH-FD had glass shard-like structures. The results indicated that the drying method could affect the physicochemical properties of hydrolysates, and SPH-SD showed potential prospects in developing functional fortified foods.

CRISPR/Cas9-mediated knockout of a prolyl-4-hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection.

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the numberof peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

Therapeutic Effect of Nile Tilapia Type II Collagen on Rigidity in CD8+ Cells by Alleviating Inflammation and Rheumatoid Arthritis in Rats by Oral Tolerance.

Fibrillins are microfibril-associated macro glycoproteins found in connective tissues and structurally related to latent TGF-β-binding proteins (LTBPs). The special cellular immunity and blocking glycoprotein receptors IIb and IIIa of fibrillins are emerging topics in recent years. In this study, Nile Tilapia type IIcollagen (NTCII) was extracted and purified from the skull cartilages by a pepsin-soluble method. Amino acid analysis indicated that NTCII consisted of 315/1000 glycine residues, 72/1000 hydroxyproline residues and 108/1000 proline residues. SDS-PAGE analysis showed that NTCII was composed of three identical 130 kDa α-chains. The results of glycoprotein/carbohydrate assay indicated that the total polysaccharide content of NTCII was 5.6–19.0%. The IR spectrum of NTCII displayed five characteristic peaks of amide I, II, III, A, B. NTCII at 10–100 μg/mL concentration downregulated the content of cytokines in the presence or absence of LPS, especially the secretion of cytokines IL-6, IL-1β and TNF-α. Interestingly, NTCII promoted the secretion of Fas/Apo-1 compared to the control group and 25 μg/mL of NTCII resulted in a higher Fas/Apo-1 secretion level in CD8+ T cells. FITC-TCII fluorescence images confirmed that NTCII could bind to the membrane surface of CD8+ T cells, leading to the induction of rigidity. NTCII could bind to the membrane surface of CD8+ T cells that leads to the induction of rigidity, as evidenced by the FITC-NTCII fluorescence images. The qRT-PCR gene expression analysis of caspase-8 collected with Fas/Apo-1 was upregulated significantly in the 1 and 50 μg/mL NTCII-treated groups compared with the control group. Overall, the results conclude that the rigidity did not lead to an increase in inflammatory factors in CD8+ T cells treated with NTCII. The oral administration of NTCII 3 mg/kg dosage caused more prominent repair of damaged ankle cartilage than the 1 mg/kg dosage in Freund’s adjuvant-induced model of arthritis in rats. Therefore, this study disclosed the immunological and anti-arthritic effect of fibrillar collagen, which could be a potential biomaterial for practical applications with lower toxicity.

Abnormal (Hydroxy)proline Deuterium Content Redefines Hydrogen Chemical Mass.

Analyzing the δ2H values in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least 4 times higher δ2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that “you are what you eat”.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

Diesel exhaust exposure in mice induces pulmonary fibrosis by TGF-β/Smad3 signaling pathway

BackgroundEpidemiological studies suggest increased risk of lung cancer associated with diesel exhaust (DE) exposure. However, DE-induced lung fibrosis may lead to cancer and needs investigation. ObjectivesTo study the mechanism involved in the initiation of DE- induced lung fibrosis. MethodsC57BL/6 mice were exposed to DE for 30 min/day for 5 days/weeks for 8 weeks. Pulmonary function test was performed to measure lung function. Mice were euthanized to collect BALF, blood, and lung tissue. BALF was used for cell count and cytokine analysis. Lung tissue slides were stained to examine structural integrity. RNA from lung tissue was used for RT-PCR. Immunoblots were performed to study fibrosis and EMT pathway. ResultsMice exposed to DE increase lung resistance and tissue elastance with decrease in inspiratory capacity (p < 0.05) suggesting lung function impairment. BALF showed significantly increased macrophages, neutrophils and monocytes (p < 0.01). Additionally, there was an increase in inflammation and alveolar wall thickening in lungs (p < 0.01) correlates with cellular infiltration. Macrophages had black soot deposition in lung tissue of DE exposed mice. Lung section staining revealed increase in mucus producing goblet cells for clearance of soot in lung. DE exposed lung showed increased collagen deposition and hydroxyproline residue (p < 0.01). Repetitive exposure of DE in mice lead to tissue remodeling in lung, demonstrated by fibrotic foci and smooth muscles. A significant increase in α-SMA and fibronectin (p < 0.05) in lung indicate progression of pulmonary fibrosis. TGF-β/Smad3 signaling was activated with increase in P-smad3 expression in DE exposed mice. Decreased expression of E-cadherin and increased vimentin (p < 0.05) in lungs of DE exposed mice indicate epithelial to mesenchymal transition. ConclusionDE exposure to mice induced lung injury and pulmonary fibrosis thereby remodeling tissue. The study demonstrates TGF-β/SMAD3 pathway involvement with an activation of EMT in DE exposed mice.

Cover Feature: Design, Synthesis, and Biochemical Evaluation of Alpha‐Amanitin Derivatives Containing Analogs of the trans‐Hydroxyproline Residue for Potential Use in Antibody‐Drug Conjugates (Chem. Eur. J. 40/2021)

Probing the hydroxyproline residue (Hyp) of alpha-amanitin, this structural activity relationship study investigates the conformation and H-bonding network of the first Hyp-analogs of the extremely toxic, RNA polymerase II inhibiting, fungal octapeptide for its potential as a warhead in antibody-drug conjugates. More information can be found in the Full Paper by A. Pahl, D. M. Perrin, et al. (DOI: 10.1002/chem.202101373).

Two \u03b2-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana

BackgroundArabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of β-glucuronosyltransferase genes/enzymes.ResultsHere, we have characterized two β-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of β-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size.ConclusionsThese results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.