Carbohydrate Side Chains Research Articles

Synthesis and Biological Evaluation of New Carbohydrate-Substituted Indenoisoquinoline Topoisomerase I Inhibitors and Improved Syntheses of the Experimental Anticancer Agents Indotecan (LMP400) and Indimitecan (LMP776)

Carbohydrate moieties were strategically transported from the indolocarbazole topoisomerase I (Top1) inhibitor class to the indenoisoquinoline system in search of structurally novel and potent Top1 inhibitors. The syntheses and biological evaluation of 20 new indenoisoquinolines glycosylated with linear and cyclic sugar moieties are reported. Aromatic ring substitution with 2,3-dimethoxy-8,9-methylenedioxy or 3-nitro groups exerted strong effects on antiproliferative and Top1 inhibitory activities. While the length of the carbohydrate side chain clearly correlated with antiproliferative activity, the relationship between stereochemistry and biological activity was less clearly defined. Twelve of the new indenoisoquinolines exhibit Top1 inhibitory activity equal to or better than that of camptothecin. An advanced synthetic intermediate from this study was also used to efficiently prepare indotecan (LMP400) and indimitecan (LMP776), two anticancer agents currently under investigation in a Phase I clinical trial at the National Institutes of Health.

Synthesis of new β2,2-amino acids with carbohydrate side chains: impact on the synthesis of peptides

The study describes the synthesis of new geminally disubstituted C-linked carbo-β2,2-amino acids (β2,2-Caas) with different carbohydrate side chains, and their use in the synthesis of β2,2-peptides. The study infers that the side chain has an influence on the synthesis of peptides and their conformational behaviour.

Glycopolymer-coated iron oxide nanoparticles: shape-controlled synthesis and cellular uptake.

Carbohydrates are involved in different cellular recognition events, and glycopolymers with carbohydrate side chains are currently being applied in many fields, with much potential for disease treatment. The aggregation shape has obvious effects on the nanoparticle-cell interaction and is therefore important for the applications of glycopolymers in biological systems. The synthesis of well-defined glyco-nanoparticles, especially non-spherical ones, is challenging work. Herein, iron oxide nanoparticles with different shapes (spindle and cubic-like) were first obtained and used as a core that was coated with dopamine methacrylamide (DMA) via catecholic chemistry for the introduction of vinyl groups. RAFT-synthesized glycopolymers were then conjugated to the DMA-coated iron oxide nanoparticles via a thiol-ene coupling reaction. By combining the convenience of inorganic nanoparticle shape control, biomimic catecholic chemistry, and efficient thiol-ene reaction, glycopolymer-decorated nanoparticles were easily obtained. Glyco-nanoparticles with variable shapes are stable in serum and exhibit shape-dependent cell uptake behaviors as well as enhanced activity toward specific lectins. The fabrication of biologically active non-spherical nanoparticles will be beneficial for both fundamental research on nanoparticle-cell interaction and related applications for disease treatment.

Influenza A virus survival in water is influenced by the origin species of the host cell

BackgroundInfluenza A viruses have an envelope made of a lipid bilayer and two surface glycoproteins, the hemagglutinin and the neuraminidase. The structure of the virus is directly dependent on the genetic makeup of the viral genome except the glycosylation moieties and the composition of the lipid bilayer. They both depend on the host cell and are in direct contact with the environment, such as air or water. Virus survival is important for virus transmission from contaminated waters in the case of wild aquatic birds or from contaminated surface or air for humans.ObjectiveThe objective of this study was to check whether the origin species of the host cell has an influence on influenza A virus survival.MethodThe persistence in water at 35°C of viruses grown on either mammalian cells or avian cells and belonging to two different subtypes H1N1 and H5N1 was compared.ResultsBoth H5N1 and H1N1 viruses remained infectious for periods of time as long as 19–25 days, respectively. However, within the same subtype, viruses grown on mammalian cells were more stable in water at 35°C than their counterparts grown on avian cells, even for viruses sharing the same genetic background.ConclusionsThis difference in virus stability outside the host is probably connected to the nature of the lipid bilayer taken from the cell or to the carbohydrate side chains of the virus surface glycoproteins. Moreover, the long-lasting survival time might have a critical role in the ecology of influenza viruses, especially for avian viruses.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Proteoglycans act as cellular hepatitis delta virus attachment receptors.

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.

On the reactivity and selectivity of donor glycosides in glycochemistry and glycobiology: trapped covalent intermediates

The reactivity of sugar donors and the stability of covalent intermediates formed in both chemical and biological systems is an active subject of study in both glycochemistry and glycobiology. Knowledge of the structure of these intermediates is vital for understanding reactivity and stereoselectivity in glycosidic bond formation, and in glycosidic bond destruction in the case of enzymatic hydrolysis. For chemical reactions, tuning of the electron-withdrawing power of the carbohydrate side chains allows for stabilization of covalent anomeric triflates thereby enabling chemo-, regio- and stereoselective glycosylations. Retaining glycosidase-mediated hydrolysis reactions in turn often involve a covalent intermediate. The existence of such covalent intermediates was convincingly demonstrated at the beginning of this century by making use of modified glycosyl substrates tuned such that stable adducts are formed efficiently but the ensuing hydrolysis is slowed down. Recently this concept has also been used in the design of glycosidase activity-based probes. This review describes recent investigations on different carbohydrate decoration patterns to influence both chemical and biological reactivity and selectivity.

Chirality and Template-Mediated Induction of Helical Preferences in Achiral β-Peptides

This study describes chirality- or template-mediated helical induction in achiral β-peptides for the first time. A strategy of end capping β-peptides derived from β-hGly (the smallest achiral β-amino acid) with a chiral β-amino acid that possesses a carbohydrate side chain (β-Caa; C-linked carbo β-amino acid) or a small, robust helical template derived from β-Caas, was adopted to investigate folding propensity. A single chiral (R)-β-Caa residue at the C- or N-terminus in these oligomers led to a preponderance of right-handed 12/10-helical folds, which was reiterated more strongly in peptides capped at both the C- and N-terminus. Likewise, the presence of a template (a 12/10-helical trimer) at both the C- and N-terminus resulted in a very robust helix. The propagation of the helical fold and its sustenance was found in a homo-oligomeric sequence with as many as seven β-hGly residues. In both cases, the induction of helicity was stronger from the N terminus, whereas an anchor at the C terminus resulted in reduced helical propensity. Although these oligomers have been theoretically predicted to favor a 12/10-mixed helix in apolar solvents, this study provides the first experimental evidence for their existence. Diastereotopicity was found in both the methylene groups of the β-hGly moieties due to chirality. Additionally, the β-hGly units have shown split behavior in the conformational space to accommodate the 12/10-helix. Thus, end capping to assist chiralty- or template-mediated helical induction and stabilization in achiral β-peptides is a very attractive strategy.

Interactions between casein layers adsorbed on hydrophobic surfaces from self consistent field theory: κ-casein versus para-κ-casein

Interactions mediated by layers of κ-casein and para-κ-casein have been calculated and compared using the self consistent field (SCF) approach, at pH and electrolyte concentrations found in milk. Our results show the interaction potentials at close separation distances to be repulsive for κ-casein, while they are attractive for para-κ-casein. We have also studied κ-casein chains stripped of their sugar moieties and highlighted the important role that the carbohydrate side chains play in the provision of steric forces. The point of transition from a repulsive to an attractive interaction is found to be rather sensitive to the degree of coverage of the surface by the protein chains. At Γ = 0.0025 chains per unit monomer area, the value is just over 40% renneted chains, whereas at Γ = 0.0032 this increases to 87%. At the coverage values higher than those we estimate for the surface of casein micelles, no transition is detected and the interactions remain repulsive even when all of κ-casein has been converted to para-κ-casein, as already shown by Mellema, Leermakers, and de Kruif (1999). At lower surface coverage values, the interaction potential curves for otherwise uncharged surfaces, covered with para-κ-casein, posses an energy barrier. We have demonstrated that the origin of this energy barrier is electrostatic, with para-κ-casein contributing a net positive charge to the surface. We have also explored the importance of the asymmetry in the distribution of charge between the para-κ-casein and the glycomacropeptide sides of the protein. The glycomacropeptide is net negative while para-κ-casein side is net positive at neutral pH. Our results suggest that on a negatively charged surface, such as the surface of casein micelles, this uneven distribution of charge is just as important in determining the conformation of κ-casein chains as is the difference in the hydrophobic and hydrophilic nature of the two sides of this protein.

Design of β-Amino Acid with Backbone–Side Chain Interactions: Stabilization of 14/15-Helix in α/β-Peptides

A new C-linked carbo-β-amino acid, (R)-β-Caa((r)), having a carbohydrate side chain with D-ribo configuration, was prepared from D-glucose by inverting the C-3 stereocenter to introduce constraints/interactions. From the NMR studies it was inferred that the new monomer may participate in additional electrostatic interactions, facilitating and enhancing novel folds in oligomeric peptides derived from it. The α/β-peptides, synthesized from alternating L-Ala and (R)-β-Caa((r)), have shown the presence of 14/15-helix by NMR (in CDCl(3), methanol-d(3) and CD(3)CN), CD and MD calculations. The hybrid peptides showed the presence of electrostatic interactions involving the intraresidue amide proton and the C3-OMe, which helped in the stabilization of the NH(i)···CO(i-4) H-bonds and adoption of 14/15-helix. The importance of such additional interactions has been well defined in recent times to stabilize the folding in a variety of peptidic foldamers. These observations suggest and emphasize that the side chain-backbone interactions are crucial in the stabilization of the desired folding propensity. The designed monomer thus enlarges the opportunities for the synthesis of peptides with novel conformations and expands the repertoire of the foldamers.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus)

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone.

Structure–Activity Relationships of α-, β1-, γ-, and δ-Tomatine and Tomatidine against Human Breast (MDA-MB-231), Gastric (KATO-III), and Prostate (PC3) Cancer Cells

Partial acid hydrolysis of the tetrasaccharide (lycotetraose) side chain of the tomato glycoalkaloid α-tomatine resulted in the formation of four products with three, two, one, and zero carbohydrate side chains, which were separated by high-performance liquid chromatography (HPLC) and identified by thin-layer chromatography (TLC) and liquid chromatography ion-trap time-of-flight mass spectrometry (LCMS-IT-TOF). The inhibitory activities in terms of IC(50) values (concentration that inhibits 50% of the cells under the test conditions) of the parent compound and the hydrolysates, isolated by preparative HPLC, against normal human liver and lung cells and human breast, gastric, and prostate cancer cells indicate that (a) the removal of sugars significantly reduced the concentration-dependent cell-inhibiting effects of the test compounds, (b) PC3 prostate cancer cells were about 10 times more susceptible to inhibition by α-tomatine than the breast and gastric cancer cells or the normal cells, (c) the activity of α-tomatine against the prostate cancer cells was 200 times greater than that of the aglycone tomatidine, and (d) the activity increased as the number of sugars on the aglycone increased, but this was only statistically significant at p < 0.05 for the normal lung Hel299 cell line. The effect of the alkaloids on tumor necrosis factor α (TNF-α) was measured in RAW264.7 macrophage cells. There was a statistically significant negative correlation between the dosage of γ- and α-tomatine and the level of TNF-α. α-Tomatine was the most effective compound at reducing TNF-α. The dietary significance of the results and future research needs are discussed.

Clinical pharmacology aspects of development and application of biosimilar antibodies

Due to the microheterogeneity of the macromolecules produced in biological systems, only the high degree of similarity but not the structural identity of the follow-on biological active agents can be proven. In case of monoclonal antibodies (mAbs) the proof of similarity is much more difficult due to the large molecular weight, complicated structure and complex biological effects of the IgG molecule. The identity of the amino acid sequence is the basic requirement of biosimilarity. Smaller changes in the carbohydrate side chains attached to the protein molecule can be accepted if they do not cause significant functional changes. On the other hand, the alteration of some carbohydrate side chain(s) located at strategically important sites might significantly influence the biological properties of the mAbs. Besides the antigen binding the pharmacological effects of the antibodies depend on several other effector functions related to the Fc fragment such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and phagocytosis. Hence several biological functions must be measured and evaluated in their totality for stating the degree of biosimilarity. According to the concordant strict requirements of the European Medicines Agency (EMA) and the American Food and Drug Administration (FDA), the proof of similar efficacy and safety must be based on the results obtained in the most sensitive and suitable in vitro and in vivo experimental designs. The fundamental proofs are provided by the comparison of the pharmacokinetic and pharmacodynamic measurements, as well as the similarity of the dose-effect relationships. These observations are complemented by the comparative clinical therapeutic trials which eventually support the similar therapeutic use of the follow-on medicinal products in the clinical practice. The final judgement of biosimilarity is a case-by-case decision based on the joint evaluation of all the data available.

Investigation of folding patterns in homo-oligomers of (R)-β2,2-amino acids with carbohydrate side chain

The study describes the synthesis of new β2,2-peptides made from geminally disubstituted β2,2-amino acid and their folding propensities. The (R)-C-linked carbo-β2,2-amino acid [(R)-β2,2-Caa] was prepared from d-glucose and converted into the homo-oligomeric di-, tetra-, and hexapeptides. The conformational studies were carried out using NMR (in CDCl3), CD, IR, and MD calculations. These β2,2-peptides were interestingly stabilized by five-membered (mr) inter-residue H-bonds NH(i)⋯O(i-1) (furanoside) and a 6-mr intra-residue H-bond between amide proton (NH(i)) and the oxygen of –OMe(i) at the C3 carbon of the carbohydrate side chain. These results amply demonstrate that the ‘epimerization’ at the spiro carbon center has an effect on the conformational behavior of these peptides. Finding of these, H-bonding patterns, which are not so common to stabilize the folds in this class of β2,2-Caa derived peptides would further facilitate augmentation in the domain of foldamer.

The pH-dependent thermal and storage stability of glycosylated caseinomacropeptide

Bioactive properties of bovine glycosylated caseinomacropeptide (gCMP) like antibacterial effects are reported to be closely associated with their structure in terms of attached glycans. Technological properties are also influenced by the carbohydrate side chains. However, during product manufacturing gCMP can be modified due to processing. Processing conditions, which influence the degree of glycosylation of gCMP lead to alterations of bioactivity and techno-functional properties of gCMP and accordingly gCMP-containing products. Hence, gCMP was studied for its glycan stability during heat treatment and storage under different pH values. Process stability (preservation of native protein structure in terms of attached glycans) was analysed by quantifying the release of the terminal carbohydrate, N-acetylneuraminic acid (Neu5Ac), from gCMP. The results clearly showed that the thermal stability of gCMP is strongly influenced by pH. When the pH was decreased from 7 to 2, reduced stability was found even at low heating temperatures. Minimal destabilisation effects were found at neutral pH. Similar observations were found during storage of gCMP. Neu5Ac was released after six days of storage, with a maximum release of 30% at pH 2. Acidic pH conditions were responsible for the hydrolysis of the glycans from the peptide backbone during heat treatment and storage.

Synthesis of C-linked carbo-β2-amino acids and β2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices

C-linked carbo-β(2)-amino acids (β(2)-Caa), a new class of β-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new β-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cβ- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-β(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The β(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on β(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the β(2)- and β(3)-peptides.

A Novel Functional Role of Collagen Glycosylation: INTERACTION WITH THE ENDOCYTIC COLLAGEN RECEPTOR uPARAP/ENDO180

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation.

Alcohol-, Diol-, and Carbohydrate-Substituted Indenoisoquinolines as Topoisomerase I Inhibitors: Investigating the Relationships Involving Stereochemistry, Hydrogen Bonding, and Biological Activity

The DNA-relaxing enzyme topoisomerase I (Top1) can be inhibited by heterocyclic compounds such as indolocarbazoles and indenoisoquinolines. Carbohydrate and hydroxyl-containing side chains are essential for the biological activity of indolocarbazoles. The current study investigated how similar functionalities could be "translated" to the indenoisoquinoline system and how stereochemistry and hydrogen bonding affect biological activity. Herein is described the preparation and assay of indenoisoquinolines substituted with short-chain alcohols, diols, and carbohydrates. Several compounds (including those derived from sugars) display potent Top1 poisoning and antiproliferative activities. The Top1 poisoning activity of diol-substituted indenoisoquinolines is dependent upon stereochemistry. Although the effect is striking, molecular modeling and docking studies do not indicate any reason for the difference in activity due to similar calculated interactions between the ligand and Top1-DNA complex and ambiguity about the binding mode. A stereochemical dependence was also observed for carbohydrate-derived indenoisoquinolines. Although similar trends were observed in other classes of Top1 inhibitors, the exact nature of this effect has yet to be elucidated.

Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

Tamm-Horsfall glycoprotein enhances PMN phagocytosis by binding to cell surface-expressed lactoferrin and cathepsin G that activates MAP kinase pathway.

The molecular basis of polymorphonuclear neutrophil (PMN) phagocytosis-enhancing activity (PEA) by human purified urinary Tamm-Horsfall glyco- protein (THP) has not been elucidated. In this study, we found human THP bound to lactoferrin (LF) and cathepsin G (CG) expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-κB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase), protein specificity (V8 protease and proteinase K) or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase). We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.