Involvement of Both the N-Glycan-relevant and N-Glycan-irrelevant Structural Elements in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.     

Non-Reducing Terminal Fucose within N-Linked Glycan Plays a Significant Role in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

We have recently demonstrated that the 1CF11 monoclonal antibody bound human milk lactoferrin (hLf) through the recognition of two distinct portions of the molecule, namely the N-glycan-relevant and -irrelevant structural elements. In this present study, we prepared four immunoreactive peptide fractions containing N-linked glycan from tryptic digests of reduced and alkylated hLf by using a concanavalin A lectin column and reverse-phase HPLC. Deglycosylation of these fractions and a competitive binding assay using fucosylated oligosaccharides revealed that the non-reducing terminal fucose residue in N-linked glycan(s) played a significant role in recognizing the N-glycan-relevant element in hLf by 1CF11.     

Structural Basis for Recognition of a Unique Epitope by a Human Anti-tau Antibody

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Unique occurrence of the 1CF11 carbohydrate epitope in primate saliva

We examined a large number of individual human and animal saliva samples for the reactivity with ICF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of ICF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with ICF11.     

人乳头瘤病毒11型L1主要衣壳蛋白的B细胞表位多肽作为疫苗的研究

分析了人乳头瘤病毒11型(HPV11)L1主要衣壳蛋白的B细胞优势表位,并以此为基础研制表位多肽疫苗。研究中采用Goldkey和．PC／Gene软件系统,分析HPV11的L1主要衣壳蛋白的二级结构、抗原性、B细胞表位,并引人氨基酸抗原性指数,综合评估其B细胞优势表位。Fmoc固相合成表位多肽,高效液相层析方法纯化,毛细管电泳分析其纯度。与0．2ml佐剂完全乳化后,按50μg／只的剂量免疫小鼠,进行动物水平的免疫效果评价。取免疫小鼠血清,与HPV11 DNA阳性的尖锐湿疣患者的疣体上清液结合,鉴定免疫后小鼠所产生抗体的特异性。发现HPV11的L1主要衣壳蛋白的第426～439位和第487～501位具有较高的免疫原性,可明显诱导小鼠血清抗体滴度升高,且该抗体与人尖锐湿疣的疣体组织上清液呈阳性反应。说明所选这两个肽段为HPV11的L1主要衣壳蛋白的B细胞优势表位,但是否具有功能特异性,尚需进一步研究。     

Studies on Tryptophane Residues in CF1-ATPase and the Enzymatic Property of CF1-ATPase Modified by NBS and Photooxidation

The numbers of tryptophane residues in spinach CF1-ATPase were measured by means of chemical modification with N-bromosuccinimide (NBS) and photooxidation. There are 3.5 tryptophane residues in CF1-ATPase, among which two are essential for the enzyme activity. Photooxidation of CF1-ATPase led to increased O2 uptake of the reaction system and loss in activity of CF1-ATPase . Immunological property of CF1-ATPase has been altered by chemical modification with NBS and photooxidation. The resuits show that tryptophane residues seen to be essential for activity and antigenic properties of CF1-ATPase.     

Epitope mapping of monoclonal antibodies for the Deinococcus radiodurans bacteriophytochome

Bacteriophytochromes (BphP) are phytochrome‐like light sensing proteins in bacteria, which use biliverdin as a chromophore. In order to study the biochemical properties of the DrBphP protein, five (2B8, 2C11, 3B2, 3D2, and 3H7) anti‐DrBphP monoclonal antibodies were produced through the immunization of mice with purified full‐length DrBphP and DrBphN (1–321 amino acid) proteins, and epitope mapping was then carried out. Among the five antibodies, 2B8 and 2C11 preferentially recognized the N‐terminal region of BphP whereas 3B2, 3D2, and 3H7 showed preference for the C‐terminal region. We performed further epitope mapping using recombinant truncated BphP proteins to narrow down their target sequences. The results demonstrated that each of the five monoclonal antibodies recognized different regions on the DrBphP protein. Additionally, epitopes of 2B8 and 3H7 antibodies were discovered to be shorter than 10 amino acids (2B8: RDPLPFFPP, 3H7: PGEIEEA). These two antibodies with such specific recognition epitopes could be especially valuable for developing new peptide tags for protein detection and purification.     

Characterization of CF1 from the Diatom Odontella sinensis

The CF₁ moiety of the chloroplast ATPase of the diatom Odontella sinensis was solubilized from isolated thylakoids by chloroform extraction. Further purification was achieved by HPLC on a Superose-6 column. The resulting four-subunit complex was identified as CF₁ lacking subunit δ. The larger two subunits, α and β, showed cross-reactivity with antisera raised against the homologous subunits of spinach-CF₁. Western blot analysis further revealed that — contrary to other ATPases — migration in SDS-PAGE of α was faster than migration of β, suggesting a deletion of 40 to 50 amino acids in subunit α of Odontella. The assumed deletion does not involve the N-terminal side of the protein, as was established by protein sequencing. The N-terminal sequences of subunits α and γ showed highest homologies with the equivalent subunits of blue-green algae. According to SDS-PAGE, the apparent molecular weights of the four Odontella subunits were 53.2 (β), 51.2 (α), 39.3 (γ) and 16.2 (ε) kD. ATPase activity of isolated Odontella-CF₁ could be induced by trypsin or octylglucoside, and to a lesser extent by sulfite or by alcohols such as methanol or ethanol.     

Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope

The monoclonal L5 antibody reacts with an N-glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na2 35SO4 and a 3H-amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine-labeled L5 proteoglycan from cultured astrocytes with proteinase-free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

Localization of the HIV-1 gp120 Conformational Epitope Recognized by Virus-Neutralizing Monoclonal Antibodies 2G12

A phage peptide library was used to select peptides interacting with virus-neutralizing monoclonal antibodies (mAb) 2G12 which recognize a discontinuous surface epitope of HIV-1 gp120. With the published X-ray data, gp120 regions involved in the antigenic determinant were predicted. Binding with mAb 2G12 was ascribed to Thr297, Phe383, Tyr384, Arg419, Ile240, Thr415, Leu416, Pro417, Lys421, and Trp112. Though distant in the gp120 sequence, these residues are close in space and form the 2G12 epitope on the gp120 surface.     

Use of the hummel and dreyer method for the study of nucleotide binding on chloroplast ATPase CF1

The study of the binding of the nucleotides ADP and ATP on the exchangeable sites of chloroplast ATPase CF1 has been carried out by the Hummel and Dreyer method applied to HPLC. It has been shown that this method was well fitted to the problem: rapidity of exchange, absence of noticeable modification after binding, presence of a constant concentration of ligand during the chromatography, which stabilizes these low affinity complexes. The dissociation constants of binding of ADP, ATP and of their magnesium salt complexes have been determined. In order to measure the simultaneous binding of ADP and ATP when present in mixture, we have modified the method by using an anion-exchange column in place of the gel filtration column: the two nucleotides were easily separated, while the binding on the protein was unchanged. The extension of this method to the reversed-phase chromatography could also be considered for the binding of hydrophobic ligands.     

Epitope characterization of an anti‐PD‐L1 antibody using orthogonal approaches

The binding of programmed death ligand 1 protein (PD‐L1) to its receptor programmed death protein 1 (PD‐1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD‐L1/PD‐1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD‐L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD‐L1. Hydrogen–deuterium exchange mass spectrometry (HDX‐MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD‐1 binding interface, consistent with a desired PD‐1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD‐L1 variants confirmed that the region identified by HDX‐MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD‐L1‐mediated immune evasion. This is the first report of an epitope for any antibody targeting PD‐L1 and demonstrates the power of combining orthogonal epitope mapping techniques. Copyright © 2015 John Wiley & Sons, Ltd.     

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.     

An Epitope of the Viscumin Catalytic Subunit Exposed on Its Interaction with Lipid Bilayer

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, p. 63). The interaction of the synthetic F¹⁰¹–T¹⁰⁵ and A⁹⁶–T¹⁰⁵ fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by ¹H NMR spectroscopy. Results of this study demonstrated that only the A⁹⁶–T¹⁰⁵ fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen–antibody complex and registered on the resonances of the free peptide transferred from the free state to the antibody-bound state was analyzed, the mab_TA7 antigen determinant (H⁹⁹–T¹⁰⁵) was identified, and its conformation and orientation within the complex with the antibody were determined.     

Epitope mapping by surface plasmon resonance in the BIAcore

An epitope may be defined as a specific site on an antigen module characterized by the binding of one monoclonal antibody (MAb). Epitope mapping by surface plasmon resonance in the BIAcore biosensor may be performed to characterize an antigen or a group of specific MAbs or both. This article describes the BIAcore instrument and methods for such mapping. Examples include molecular interaction studies with simple and complex proteins, such as myoglobin and calprotectin, respectively.     

抗HLJ1单克隆抗体的制备及抗原检测方法的建立

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。     

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11‐derived dihydrofolate reductase deficient host cell

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR‐deficient DG44, and DUXB11‐based DHFR deficient CHO. Current Genentech commercial full‐length antibody products have all been produced in the DUXB11‐derived DHFR‐deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11‐derived DHFR‐deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11‐based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14‐day fed batch cultures in shake flasks. In contrast, the DUXB11‐based host produced ～0.1 g/l for both antibodies in the same 14‐day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ～2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:980–985, 2013     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.     

Ovalbumin-Related Gene Y Protein Bears Carbohydrate Chains of the Ovomucoid Type

We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490–2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid.