Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides

A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attached N-acetyl-D-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated O-linked glycopeptides should be due to the trimer formation of rHML.     

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.     

Mucin core O-glycosylation is modulated by neighboring residue glycosylation status. Kinetic modeling of the site-specific glycosylation of the apo-porcine submaxillary mucin tandem repeat by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases T1 and T2

The influence of peptide sequence and environment on the initiation and elongation of mucin O-glycosylation is not well understood. The in vivo glycosylation pattern of the porcine submaxillary gland mucin (PSM) tandem repeat containing 31 O-glycosylation sites (Gerken, T. A., Gilmore, M., and Zhang, J. (2002) J. Biol. Chem. 277, 7736-7751) reveals a weak inverse correlation with hydroxyamino acid density (and by inference the density of glycosylation) with the extent of GalNAc glycosylation and core-1 substitution. We now report the time course of the in vitro glycosylation of the apoPSM tandem repeat by recombinant UDP-GalNAc:polypeptide alpha-GalNAc transferases (ppGalNAc transferase) T1 and T2 that confirm these findings. A wide range of glycosylation rates are found, with several residues showing apparent plateaus in glycosylation. An adjustable kinetic model that reduces the first-order rate constants proportional to neighboring glycosylation status, plus or minus three residues of the site of glycosylation, was found to reasonably reproduce the experimental rate data for both transferases, including apparent plateaus in glycosylation. The unique, transferase-specific, positional weighting constants reveal information on the peptide/glycopeptide recognition site for each transferase. Both transferases displayed high sensitivities to neighboring Ser/Thr glycosylation, whereas ppGalNAc T2 displayed additional high sensitivities to the presence of nonglycosylated Ser/Thr residues. This is the first demonstration of the ability to model mucin O-glycosylation kinetics, confirming that under the appropriate conditions neighboring glycosylation status can be a significant factor modulating the first step of mucin O-glycan biosynthesis.     

O-glycosylation in Toxoplasma gondii: identification and analysis of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

The initiation of mucin-type O-glycosylation is catalysed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (EC 2.4.1.41). These enzymes are responsible for the transfer of N-acetylgalactosamine from the nucleotide sugar donor, UDP-GalNAc, to the hydroxyl group on specific serine or threonine residues in acceptor proteins. By screening a Toxoplasma gondii cDNA library, three distinct isoforms of the ppGalNAc-T gene family were cloned. Two additional isoforms were identified and partially cloned following analysis of the T. gondii genome sequence database. All of the cloned and identified ppGalNAc-T's are type II membrane proteins that share up to 50% amino acid sequence identity within the conserved catalytic domain. They each contain an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a lumenal domain; the latter consists of stem, catalytic, and lectin-like domains. Moreover, each of this ppGalNAc-T's contains important sequence motifs that are typical for this class of glycosyltransferases. These include a glycosyltransferase 1 motif containing the DXH sequence, a Gal/GalNAc-T motif, and the CLD and QXW sequence motifs located in alpha-, beta-, and gamma-repeats present within the lectin-like domain. The coding regions of T. gondii ppGalNAc-T1, -T2, and -T3 reside in multiple exons ranging in number from 6 to 10. Our results demonstrate that mucin-type O-glycosylation in T. gondii is catalysed by a multimember gene family, which is evolutionarily conserved from single-celled eukaryotes through nematodes and insects up to mammals. Taken together, this information creates the basis for future studies of the function of the ppGalNAc-T gene family in the pathobiology of this apicomplexan parasite.     

Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.     

Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide

BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Evidence for dopaminergic modulation of pancreatic polypeptide secretion in man

This study examines the role of dopaminergic mechanisms in the regulation of human pancreatic polypeptide (hPP) secretion in 11 normal male volunteers. Administration of domperidone (20 mg iv), an extracerebral inhibitor of dopamine receptors, resulted in a hPP rise (p<0.05) within 10 min and a peak response (p<0.01) at 15 min after drug administration. Administration of the dopaminergic agonist, bromocriptine, 2.5 mg tid for 4 days eliminated hPP responses to isometric handgrip exercise in these 11 volunteers. These results suggest that dopaminergic mechanisms may exert a tonic inhibitory effect on hPP secretion in normal subjects.     

Syntheses and biological activities of atrial natriuretic polypeptide analogs

Recently several peptides with natriuretic and diuretic potencies were isolated from human and rat atrial extract, and the precursors of the peptides were sequenced. Of the peptides, -human and rat atrial natriuretic polypeptides (-hANP, -rANP), consisting of 28 amino acids, are thought to be essential to the potency and to play an important role in the blood pressure regulation system. The amino acid sequence of -hANP is different from that of -rANP only at the position 12 (isoleucine in -rANP). In the present study, we synthesized ANPs and their analogs using a new deprotection procedure based on the concept of push-pull mechanism. Using the synthetic ANP analog, we also developed a radioimmunoassay for -ANP and examined the structure-activity relationship. Synthetic -hANP caused potent, rapid, and short-acting increases in Na⁺ and Cl^– excretion, and also an increase in urine flow and K⁺ excretion of lesser magnitude, when injected into rat. Also, we synthesized a cyclic part of -hANP, -ANP(7–23)-NH₂. Since this peptide had a little diuretic and natriuretic potency, we attempted to synthesize a chemically stable -hANP analog. We considered that the disulfide bond would be equivalent to propylene with regard to interatomic distance and employed 8-aminocaprylic acid instead of cystine. This cyclic peptide, named cyclonatrin-54, had a somewhat higher potency than -hANP(7–23)-NH₂ for diuresis and natriuresis, as expected. Furthermore, using a synthetic intermediate of cyclonatrin-54, we prepared a linear ANP analog, -hANP(8–22), Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly. This linear 15-amino acid peptide had a dose-dependent natriuretic and diuretic activity, but no hypotensive effect. It was surprising that a linear peptide exhibited a potent natriuretic activity. For the first time, a linear peptide has been prepared that has substantial natriuretic and diuretic potency. We synthesized some analogs of this 15-amino acid peptide and investigated the structure-activity relationship.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium

O₂ regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O₂-threshold for culmination implicating it as a direct O₂-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3^SCFubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O₂-signal. Here we show that genetically induced changes in Skp1 levels also affect the O₂-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3^SCFubiquitin-ligase activity modulate O₂-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O₂-sensing.     

ATP binding and ATPase activities associated with recombinant rabbit hemorrhagic disease virus 2C-like polypeptide

The carboxy-terminal region of the rabbit hemorrhagic disease virus p37 polyprotein cleavage product has been expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant GST-Delta2C protein showed in vitro ATP-binding and ATPase activities. Site-directed mutagenesis studies of the conserved residues G(522) and T(529) in motif A, D(566) and E(567) in motif B, and K(600) in motif C were also performed. These results provide the first experimental characterization of a 2C-like ATPase activity in a member of the Caliciviridae.     

TGFβ regulation of membrane mucin Muc4 via proteosome degradation

Muc4 is a heterodimeric membrane mucin implicated in epithelial differentiation and tumor progression. It is expressed from a single gene as a 300 kDa precursor protein which is cleaved in the endoplasmic reticulum to its two subunits. Our previous work has shown that Muc4 is regulated by TGFβ, which represses the precursor cleavage. Working with Muc4‐transfected A375 tumor cells, we now show that Muc4 undergoes proteosomal degradation. Proteosome inhibitors prolong the life of the precursor, shunt the Muc4 into cytoplasmic aggresomes, increase the level of Muc4 associated with the endoplasmic reticulum chaperones calnexin and calreticulin and increase the levels of ubiquitinated Muc4. Most importantly, proteosome inhibitors repress the TGFβ inhibition of Muc4 expression. These results suggest a model in which TGFβ inhibits precursor cleavage, shunting the precursor into the proteosomal degradation pathway. Thus, the cells have evolved a mechanism to use the quality control pathway for glycoproteins to control the quantity of the protein produced. J. Cell. Biochem. 107: 797–802, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence for a C4b binding site on the C2b domain of C2

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.     

Evidence for distinct kynureninase and hydroxykynureninase activities in Neurospora crassa

Previous studies have indicated that a single enzyme, "kynureninase," catalyzes the reactions of l-kynurenine to anthranilate and l-3-hydroxykynurenine to 3-hydroxyanthranilate in Neurospora crassa and in other organisms. The present report describes separate enzymes which catalyze these reactions in N. crassa. The first, a kynureninase, preferentially catalyzes kynurenine to anthranilate and is induced over 400-fold by tryptophan or a catabolite of tryptophan. The second, a hydroxykynureninase, is constitutive or noninducible by tryptophan and preferentially catalyzes l-3-hydroxykynurenine to 3-hydroxyanthranilate. The physiological significance of these enzymes may be inferred from the facts that (i) the noninducible enzyme hydroxykynureninase appears to be the main enzyme present in uninduced cells that is capable of catalyzing l-3-hydroxykynurenine to 3-hydroxyanthranilate for the indispensible synthesis of nicotinamide adenine dinucleotide, and (ii) the inducible enzyme kynureninase is induced by tryptophan to a concentration far in excess of that needed to meet the requirements of the cells for nicotinamide adenine dinucleotide, resulting in the excretion of anthranilate into the medium.     

Effect of Cd and UV-B radiation on polypeptide composition and photosystem activities ofVigna unguiculata chloroplasts

Rates of whole chain and photosystem 2 activities in chloroplasts isolated fromVigna unguiculata L. seedlings grown under ultraviolet-B (UV-B) enhanced radiation were less affected by 3, 6 and 9 mM CdCl₂ for 60 min at 0 °C in the dark than the rates in chloroplasts from control plants grown under normal irradiation. The results are in agreement with changes in contents of chloroplast 55, 47, 43, 33, 29, 27–25, 23 and 17 kDa polypeptides that were significantly lowered at 3, 6 and 9 mM CdCl₂ only in chloroplasts from control plants. On the other hand, in the simultaneous treatment of chloroplast isolated from control plants the UV-B supported the inhibitory effect of all applied concentrations of CdCl₂. The photosystem 1 activity was only marginally affected in the all experimental variants.     

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.     

Synthesis and accumulation of mannose-containing glycopeptides in human fibroblast cells possible separate pathways for neutral and acidic glycopeptides

The normal human fibroblast, WI-38, was labelled with radioactive mannose and its incorporation, as well as the accumulation of acidic and neutral glycopeptides on the cell surface, was followed as a function of time. The transit time of newly made Pronase-released cell surface glycopeptides from their intracellular site of synthesis to the cell surface was slower in nongrowing cells than in a rapidly growing culture. When the surface glycopeptides were separated by high-voltage paper electrophoresis into neutral and acidic species, it was observed that the cell surface material was initially enriched with neutral glycopeptides. However, with time the relative proportion of acidic species increased so that by 3 h the ratio between the acidica and neutral species approached a constant value. Our data are consistent with the hypothesis that multiple pathways for asparagine-linked glycoprotein biosynthesis are possible.