Biology subjects

Chapla, D.

Publications and source records attributed to Chapla, D..

9 recordsLinked to original sources

bioRxiv2026

Identification of glycosyltransferases that synthesize pectin heteroglycan block polymers and a structural motif required for their activity

Pectin is a family of plant cell wall complex polysaccharides and proteoglycans comprised of the glycan domains homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted HGs rhamnogalacturonan II (RG-II), xylogalacturonan and apiogalacturonan. The number, type, and arrangement of the pectic domains in different pectic polymers are unknown, as is the identity of the enzymes that link the domains together. Here we show that two Arabidopsis -1,4-galacturonosyltransferases, GAUT13 and GAUT14, in addition to previously reported HG acceptor-dependent and HG de novo synthesis activities, initiate and elongate HG on the non-reducing end of RG-I, identifying enzymes that synthesize pectic RG-I:HG heteroglycans. While other catalytic GAUTs did not initiate HG on RG-I acceptors, they were able to elongate HG once initiated on RG-I by GAUT13 and GAUT14. We show that the three different GAUT14 acceptors (UDP-GalA, HG, and RG-I) compete for the same binding pocket, and amino acids required for GAUT14 catalytic activity were identified using in vitro enzyme assays, computational modeling, and site-directed mutagenesis. The transfer of the GAUT14 hypervariable 1 (HV1) motif (RTFASKLQSRS) was sufficient and necessary to transfer RG-I:HG extension activity to GAUT10, which otherwise does not have this activity, representing a novel functional glycosyltransferase domain swapping strategy. The presence of all three catalytic activities in a GAUT13/14 homolog from the non-vascular seedless plant Physcomitrium established the early evolution of GAUT13/14 trifunctionality. The identification of GAUT13 and GAUT14 as RG-I:HG extension catalysts provides targets for probing in planta structure and function of RG-I:HG heteropolymers.

plant biology↗

bioRxiv2026

CRISPR activation screens identify core protein-dependent regulation of heparan sulfate sulfation and ligand specificity

Heparan sulfate proteoglycans (HSPGs) are essential cell surface and extracellular matrix glycoconjugates that mediate diverse biological processes through interactions between their heparan sulfate (HS) chains and extracellular ligands. While HS sulfation patterning is known to dictate ligand specificity, how cells control HS assembly to regulate these interactions remains incompletely understood. To systematically identify genetic modifiers of HS-protein interactions, we performed genome-wide CRISPR activation (CRISPRa) screens in HEK293T cells using binding of antithrombin (AT), which selectively recognizes 3-O-sulfated HS motifs, or the N-sulfation-specific antibody 10E4 as functional readouts. Strikingly, the screens revealed proteoglycan core proteins as key modulators of HS function. In particular, syndecan-1 (SDC1) emerged as a preferential enhancer of AT binding compared to other syndecan family members. Targeted upregulation of syndecan family members increased total HS levels, but only SDC1 enhanced AT binding. Structural and enzymatic analyses demonstrated that SDC1-associated HS chains contain elevated 6-O-sulfation and serve as superior substrates for 3-O-sulfotransferases relative to SDC2-associated HS chains. Additionally, SDC1 exhibited slower cell surface recovery, which was blocked by cycloheximide treatment, consistent with extended trafficking and biosynthetic processing. Overall, these findings indicate that proteoglycan core protein identity influences HS sulfation patterning and ligand-binding specificity and trafficking kinetics may contribute to core protein-dependent regulation of HS modification.

Cell Biology↗

bioRxiv2025

Mucin-type O-glycans regulate proteoglycan stability and chondrocyte maturation

O-glycosylation is a ubiquitous post-translational modification essential for protein stability, cell signaling, and tissue organization, yet how distinct O-glycan subclasses coordinate tissue development remains unclear. Here, we identify functional crosstalk between extended mucin-type O-glycans and heparan sulfate proteoglycans (HSPGs). Genetic ablation of {beta}-1,3-galactosyltransferase 1 (C1GALT1) or its chaperone COSMC in human chondrocytes reduced cell surface HSPGs and fibroblast growth factor (FGF) binding, leading to impaired MAPK/ERK signaling, which was recapitulated in hypomorphic COSMC-CDG patient cells. Transcriptomic and secretome analyses revealed selective loss of proteoglycan expression and broader extracellular matrix remodeling in COSMC- and C1GALT1-deficient cells. Mechanistically, truncation of O-GalNAc glycans reduced Syndecan-1 levels at the cell surface via enhancing its lysosome-dependent degradation and diminished CD44v3-mediated FGF1 binding. Functionally, loss of complex O-GalNAc glycans disrupted chondrogenesis of growth plate-like chondroprogenitors. Overall, these findings reveal previously unrecognized roles for mucin-type O-glycans in maintaining proteoglycan stability and function, highlighting cross-regulatory mechanisms of glycosylation crucial for normal development that likely contribute to the pathophysiology of glycosylation-related disorders.

cell biology↗

bioRxiv2025

Biochemical and structural characterization of glycosyltransferase family 61 proteins reveal a key determinant of sugar donor specificity

Xylan, the most abundant non-cellulosic polymer in plant cell walls, is structurally diverse, especially in grasses where it is heavily substituted with arabinofuranose and further modified by various residues. Common substitutions across species include glucuronic and 4-O-methyl-glucuronic acid. Arabinose and xylose sidechains are synthesized by glycosyltransferase family 61 (GT61) proteins, many of which remain uncharacterized in plants, with limited structural and mechanistic understanding. In this study, we identified two novel GT61 enzymes in Sorghum bicolor, functioning as xylan arabinosyltransferase (SbXAT) and xylan xylosyltransferase (SbXXT). We resolved the crystal structure of SbXAT, which exhibits a GT-B fold with two Rossmann-like domains linked by a cleft that accommodates the catalytic site. Structural comparison with a predicted SbXXT model revealed a substrate-binding residue critical for sugar donor specificity, validated through site-directed mutagenesis and enzymatic assays. These findings enhance understanding of xylan biosynthesis and provide a foundation for engineering glycosyltransferases and predicting their functions.

biochemistry↗

bioRxiv2023

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to prepare well-defined KS oligosaccharides by exploiting known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfates terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify internal galactosides of a poly-LacNAc chain, however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also presentation of epitopes as part of an O- or N-glycan determines binding properties.

biochemistry↗

bioRxiv2023

The Plasminogen-Apple-Nematode (PAN) domain suppresses JA/ET defense pathways in plants

Suppression of immune response is a phenomenon that enables biological processes such as gamete fertilization, cell growth, cell proliferation, endophyte recruitment, parasitism, and pathogenesis. Here, we show for the first time that the Plasminogen-Apple-Nematode (PAN) domain present in G-type lectin receptor-like kinases is essential for immunosuppression in plants. Defense pathways involving jasmonic acid and ethylene are critical for plant immunity against microbes, necrotrophic pathogens, parasites, and insects. Using two Salix purpurea G-type lectin receptor kinases, we demonstrated that intact PAN domains suppress jasmonic acid and ethylene signaling in Arabidopsis and tobacco. Variants of the same receptors with mutated residues in this domain could trigger induction of both defense pathways. Assessment of signaling processes revealed significant differences between receptors with intact and mutated PAN domain in MAPK phosphorylation, global transcriptional reprogramming, induction of downstream signaling components, hormone biosynthesis and resistance to Botrytis cinerea. Further, we demonstrated that the domain is required for oligomerization, ubiquitination, and proteolytic degradation of these receptors. These processes were completely disrupted when conserved residues in the domain were mutated. Additionally, we have tested the hypothesis in recently characterized Arabidopsis mutant which has predicted PAN domain and negatively regulates plant immunity against root nematodes. ern1.1 mutant complemented with mutated PAN shows triggered immune response with elevated WRKY33 expression, hyperphosphorylation of MAPK and resistant to necrotrophic fungus Botrytis cinerea. Collectively, our results suggest that ubiquitination and proteolytic degradation mediated by the PAN domain plays a role in receptor turn-over to suppress jasmonic acid and ethylene defense signaling in plants.

plant biology↗

bioRxiv2023

Bacterial mixed cultures followed mixed-order kinetics during mono-azo dye decolourization and degradation in batch studies

Over the years little has been focused on kinetic and thermodynamic aspect of bioremediation studies on dyes. In this study, detailed kinetic and thermodynamic analyses of results obtained during a primary study on a mono-azo dye: Reactive violet 5R (RV5), decolourization and degradation by bacterial mixed cultures SB4 were performed. Various kinetic models (zero-, first- and second-order behaviour and Monod model) were applied to understand the decolourization mechanism in batch process. Similarly, steady-state kinetics for determination of Michaelis-constant and maximal dye removal rate was derived. With increasing RV5 concentration, the specific decolourization rate had increased from 0.61mg/L/h to 1.38mg/L/h. The apparent kinetics of RV5 decolourization reaction followed mixed-order behaviour. The process was endothermic. The enthalpy and entropy values calculated to 45.6kJ/mol and 146J/mol/K, respectively, with an activation energy of 87kJ/mol. The Gibbs free energy change suggests that, decolourization reaction under experimental conditions was non-spontaneous below 20{degrees}C but was spontaneous at higher temperatures.

microbiology↗

bioRxiv2022

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

N-glycosylation is an essential eukaryotic post-translational modification that affects various glycoprotein properties, including folding, solubility, protein-protein interactions, and half-life. N-glycans are processed in the secretory pathway to form varied ensembles of structures, and diversity at a single site on a glycoprotein is termed microheterogeneity. To understand the factors that influence glycan microheterogeneity, we hypothesized that local steric and electrostatic factors surrounding each site influences glycan availability to enzymatic modification. We tested this hypothesis by expression of a panel of reporter N-linked glycoproteins in MGAT1- null HEK293 cells to produce immature Man5GlcNAc2 glycoforms (38 glycan sites total). These glycoproteins were then sequentially modified in vitro from high-mannose to hybrid and on to biantennary, core fucosylated, complex structures by a panel of N-glycosylation enzymes and each reaction time-course was quantified by LC-MS/MS. Substantial differences in rates of in vitro enzymatic modification were observed between glycan sites on the same protein and differences in modification rates varied depending on the glycoenzyme being evaluated. By comparison, proteolytic digestion of the reporters prior to N-glycan processing eliminated differences in in vitro enzymatic modification. Comparison of in vitro rates of enzymatic modification with the glycan structures found on the mature reporters expressed in wild type cells correlate well with the enzymatic bottlenecks found in vitro. These data suggest that higher-order local structures surrounding each glycosylation site contribute to the efficiency of modification both in vitro and in vivo to establish the spectrum of site-specific microheterogeneity found on N-linked glycoproteins.

biochemistry↗

bioRxiv2020

Glycan remodeled erythrocytes facilitate antigenic characterizationof recent A/H3N2 influenza viruses

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. We examined receptor specificities of A/H3N2 viruses, revealing that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing 2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of several antigenically different viruses in humans. Computational studies of HAs in complex with a sialoside having an extended LacNAc moiety revealed that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site.

biochemistry↗