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Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.

Background Decaarginine-polyethylene glycol-conjugated 3,5-bis(dodecyloxy)benzamide/plasmid DNA [Arg10-polyethylene glycol (PEG)-lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated. Methods R10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µm or 25 µm R10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light-electron microscopy (CLEM). Results EM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B-mediated gene delivery. Conclusions We have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B-mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine-mediated carriers. Copyright © 2012 John Wiley & Sons, Ltd.

The supramolecular organogelation of alcohols was observed in relatively hydrophobic amphiphiles with a short oligo(ethylene glycol) unit and three long alkyl chains at room temperature, while the hydrogelation occurred in more hydrophilic gelators with a longer poly(ethylene glycol) unit and two long alkyl chains at various temperatures. When a hot aqueous solution of some of the synthetic hydrogelators was cooled down, the supramolecular hydrogel was formed at room temperature. In some other amphiphiles with less intermolecular interactivity in water at room temperature, a reverse phase transition of sol to gel was observed by elevating the temperature of their aqueous systems, especially below a physiological temperature, 37 °C. The supramolecular hydrogelation at a low or high temperature was dependent on a slight molecular modification of the synthetic amphiphiles.

A luminescent hydrogel was successfully prepared by immobilizing an europium(III) tetrakis(β-diketonate) complex into a gallate-based hydrogelator. The Eu(III) emission in hydrogel media was switched reversibly “on-off” as a function of pH and the corresponding thermal and photostabilities dramatically increased compared with its solution sample.

Fullerene derivative C60TT, which is substituted with the low-molecular-weight organogelator tris(dodecyloxy)benzamide, formed nanowire structures on application of the Langmuir-Blodgett (LB) method. The surface morphology of the C60TT LB film was dependent on the holding time before deposition at a surface pressure of 5 mN m(-1); it changed from a homogeneous monolayer to a bilayer fibrous structure via a fibrous monolayer structure, which was estimated to have dimensions of 1.2 nm in height, 8 nm in width, and 5-10 microm in length. From the structural and spectroscopic data, it is inferred that close packing of the fullerene moiety occurs along with intermolecular hydrogen bonding within the monolayer fibrous structure. The morphological changes in the LB film are explained kinetically by the Avrami theory, based on the decrease in the surface area of the monolayer at the air/water interface. The growth of the quasi-one-dimensional fibrous monolayer structures at holding times from 0 to 0.2 h is considered to be an interface-controlled process, whereas the growth of the quasi-one-dimensional bilayer fibrous structures from 0.2 to 18 h is thought to be a diffusion-controlled process.

We numerically demonstrate spatiotemporal control of ultrafast localized plasmon pulses at metallic nanostructures. This results confirm that more flexible spatiotemporal control can be achieved by femtosecond laser pulses combined with wave front and pulse shaping.

Spatiotemporal nanofocusing of surface plasmon polaritons excited by femtosecond laser pulses on a tapered metallic tip with few tens nanometer radius is deterministically controlled using a measured plasmon response function.

Nanofocusing of ultrafast surface plasmon polariton pulses excited on an Au tapered tip, and the nonlinear radiation such as second harmonics and coherent anti-stokes Raman scattering are deterministically controlled based on a measured plasmon response function.

The Langmuir−Blodgett (LB) method was applied to low molecular weight organogelator N-[N-(6-hydroxyhexyl)-3-carbamoylpropyl]-3,4,5-tris(dodecyloxy)benzamide (1) to form molecular-assembly fibers on a solid substrate. Molecule 1 formed a stable monolayer up to 40 mN/m at the air−water interface. The monolayer was transferred onto a mica surface at 35 mN/m. In the transferred film, fibrous structures with typical dimensions of 1.2 nm (height) × 50−100 nm (width) × 5−10 μm (length) were observed by atomic force microscopy. These structures were ordered in specific directions, reflecting the C6 surface crystal symmetry of mica. Lateral modulation frictional force microscope measurements revealed that the fibrous structures had high crystallinity and that the surface groups were hydrophilic. These results enabled us to construct functional fibers by introducing functional units into the hydrophilic part of molecule 1. N-[N-(6-Aminohexyl)-3-carbamoylpropyl]-3,4,5-tris(dodecyloxy)benzamide hydrochloride (2) has ...

As models of naturally occurring glycolipids, structurally well-determined amphiphilic compounds were prepared. The synthetic molecules have β- d -galactopyranosyl or α- d -mannopyranosyl and two dodecyl groups as terminal hydrophilic sugar and hydrophobic hydrocarbon moieties, respectively. The two long alkyl chains are connected by 3,5-dioxybenzamide through ether linkages to give a lipid analog purified easily due to its absorbance of ultraviolet light. In the synthetic glycolipids, the glycoside and lipid parts are covalently bound via an oligomethylene spacer. The glycolipids could be easily incorporated into liposomes of l -α-phosphatidylcholine. The monoglycosyl moiety of the synthetic glycolipids possessing a hexamethylene spacer was present on the surface of the liposomes and interacted specifically with a lectin to give liposomal assemblies. Such agglutination of these liposomes induced by lectins was determined by analyses of turbidity and particle size based on dynamic light scattering and laser diffraction methods. The other liposomes possessing a shorter ethylene or longer decamethylene linker gave few lectin-induced agglutinates, indicating that these spacers were not effective for the presentation of the galacto-terminal on the liposomal surfaces. Similar spacer-dependent recognition of ricin with a galactolipid-incorporated phospholipid monolayer was confirmed by surface plasmon resonance technique on a substrate.

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N -acetyl- d -glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo -β- N -acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo -β- N -acetylglucosaminidase from Arthrobacter protophormiae . Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.

Purpose: To establish a novel formulation tumor-targeted drug carrier of lipophilic antitumor antibiotics, aclacinomycin A (ACM), folate-linked microemulsions were prepared and investigated both in vitro and in vivo. Experimental Design: Three kinds of folate-linked microemulsions with different polyethylene glycol (PEG) chain lengths loading ACM were formulated with 0.24 mol% folate-PEG2000-distearoylphosphatidylethanolamine (DSPE), folate-PEG5000-DSPE, and folate-lipid (without PEG linker) in microemulsions. In vitro studies were done in a human nasopharyngeal cell line, KB, which overexpresses the folate receptor (FR), and a human hepatoblastoma cell line, [FR(−)] HepG2. In vivo experiments were done in a KB xenograft by systemic administration of folate-linked microemulsions loading ACM. Results: The association of folate-linked microemulsions to KB cells could be blocked by 2 mmol/L free folic acid. Selective FR-mediated cytotoxicity of folate-linked microemulsions loading ACM was obtained in KB but not in HepG2 cells. The association of the folate-PEG5000-linked microemulsion and folate-PEG2000-linked microemulsion with the cells was 200- and 4-fold higher, whereas their cytotoxicity was 90- and 3.5-fold higher than those of nonfolate microemulsion, respectively. The folate-PEG5000-linked microemulsions showed 2.6-fold higher accumulation in solid tumors 24 hours after i.v. injection and greater tumor growth inhibition than free ACM. Conclusion: These findings suggest that a folate-linked microemulsion is feasible for tumor-targeted ACM delivery. This study shows that folate modification with a sufficiently long PEG chain on emulsions is an effective way of targeting emulsion to tumor cells.

糖鎖の付加がペプチド主鎖の立体構造にどのような影響を与えるかという根本的な課題は、この十数年程の糖ペプチドの合成の進歩と詳細なNMR解析によって、基本的な見通しを得たように思える。即ち、糖鎖の付加は糖鎖付加部位周辺の糖鎖部分とペプチド部分との相互作用によってペプチド主鎖の立体構造に何らかの影響を与える。その影響の程度は、解析的手法による検出が容易な、糖鎖とペプチドの特定原子間の接触による局所的な立体構造の変化から、解析的手法による検出が困難な、個別の原子間接触が特定できない、糖鎖の排除体積効果によるわずかな配座空間の変化まで、見た目の違いはあるものの、糖鎖が結合したことによってペプチド主鎖の配座に変化が起こるという点に関しては共通して理解することができる。見た目の立体配座の変化の違いは、糖鎖が結合しているアミノ酸残基の構造、糖鎖構造、糖鎖が結合する位置あるいはその周囲のアミノ酸配列等に影響を受ける。糖鎖とペプチドの間の遠距離の相互作用の有無は、ペプチドあるいはタンパク質の形成するさらに大きな立体構造に依存する。以上に述べた、糖鎖がペプチド主鎖の立体構造に与える影響に関する統一的な捉え方に従って、我々が研究した糖鎖付加カルシトニンの結果を振り返ってみたい。

Starting from N-glycosylated eel calcitonin derivatives that contain an N-acetyl-D-glucosamine residue specifically at the 3rd, 14th, 20th or 26th amino acid residue, corresponding glycopeptides with a complex-type oligosaccharide attached to the respective amino acid residue were synthesized by means of a transglycosylation reaction catalyzed by an endo-β-N-acetylglucosaminidase from Mucor hiemalis. The use of a recombinant enzyme and an excess of a glycosyl donor led to a yield in excess of 60%. Calcitonin derivatives containing truncated oligosaccharides were also prepared via digestion of the complex-type N-glycan with exoglycosidases. Using these N-glycosylated calcitonin derivatives, the effect of carbohydrate structure and glycosylation site on the three-dimensional structure and the biological activity of the peptide were studied. The conformation of the peptide backbone did not change irrespective of the carbohydrate structure or the glycosylation site. However, hypocalcemic activity, calcitonin-receptor binding activity and the biodistribution of the derivatives were affected by the glycosylation and were dependent on both the carbohydrate structure and the glycosylation site. Although the larger oligosaccharides tended to hinder receptor binding, the biodistribution altered by N-glycosylation appeared to enhance the hypocalcemic activity in some cases, and the magnitude of the effect was dependent on the site of glycosylation. Published in 2003.

Cell growth and production of interferon-β (IFN-β) were investigated for normal human skin fibroblast cells (NB1-RGB) cultured on membranes prepared from peptide-lipids containing the arginine-glycine-aspartic acid [Arg-Gly-Asp] (RGD), tyrosine-isoleucine-glycine-serine-arginine [Tyr-Ile-Gly-Ser-Arg] (YIGSR) and arginine-glutamic acid-aspartic acid-valine [Arg-Glu-Asp-Val] (REDV) peptides. Cell density was found to be approximately the same on various peptide-lipid membranes, whereas production of IFN-β depended significantly on the peptide-lipid membranes on which NB1-RGB cells were cultured. The highest production of IFN-β was observed for NB1-RGB cells on REDV-lipid membranes prepared by a casting method (REDV-cast membranes) after 24 hr of cultivation. Specific binding between REDV of REDV-cast membranes and the receptor on the NB1-RGB cells may have caused the specific cell response for the production of IFN-β.

We previously reported that two out of seven artificially O-glycosylated calcitonin derivatives had an altered peptide backbone conformation as indicated by decreased helical contents, determined by CD measurement. In the present study, two of those derivatives, in which a GalNAc residue is attached to Thr6 or Thr21 of calcitonin, were analyzed by NMR in order to determine the structural changes induced by the O-glycosylation in more detail. Deviations in the chemical shifts suggest that the structural change is not global but only a local one and is located in the vicinity of each O-glycosylation site. The intensities of the NOE cross peaks, an indicator of α-helical structure, also were decreased around the O-glycosylation site. The hydrogen/deuterium exchange rates of the main chain amide protons increased at the N- or C-terminal portion of the α-helix corresponding to the respective O-glycosylation site and explains the results of the CD experiments. The inter-residual NOE cross peaks between the carbohydrate and the peptide portions, other than the O-glycosylated amino acid residue, showed that local structural contacts extended three or two residue distance for Thr6- or Thr21-glycosylated derivative, respectively. Thus, we conclude that the O-glycosylation induced a change in the local structure and that this structural perturbation modulated the original α-helical structure of calcitonin, resulting in the apparent decrease in the helical content deduced from CD spectra.

Two types of poly(ethylene glycol) (PEG)-based amphiphiles were synthesized: a diblock-type amphiphile (PEG1) with a hydrophobic moiety on one terminal of PEG (PEG with the molecular weight of 2000), and a triblock-type amphiphile (PEG2) with hydrophobic moieties on both terminals of PEG. The hydrophobic moiety consisted of hydrophobic C14 alkyl tails covalently linked through a gallamide component. The sol–gel transitions of the aqueous solution of PEG1 and PEG2 were studied. At the PEG amphiphile concentration of 15 wt %, PEG1 gave a clear solution over a wide temperature range from 20 to 80 °C, while PEG2 was partially soluble in water. However, the aqueous blend solution of PEG1 and PEG2 exhibited a hydrogel depending on the blend ratio and temperature. It was considered that the composition of PEG1 and PEG2 governed the sol–gel transition, and that the dynamic moduli could be effectively controlled by changing the mixing ratios of the PEG amphiphiles. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45402.

Spatiotemporal nanofocusing of ultrafast surface plasmon polaritons (SPPs) coupled on a metal Au tapered tip with a curvature radius of a few tens of nanometers is deterministically controlled based on the measured plasmon response function. We control the SPP pulse shape and the second harmonic generation at the apex of the Au tapered tip by shaping the excitation femtosecond laser pulses based on the response function. We also adapted a similar control scheme for coherent anti-Stokes Raman scattering (CARS) and achieved selective CARS excitation of a single Raman mode of carbon nanotubes with only a single excitation laser pulse at the apex of the tip.

Coating a dish for suspension culture with 3,4,5-tris(dodecyloxy)benzyl alcohol (TDOB-OH), TDOB glucoside (TDOB-Glc), or TDOB galactoside (TDOB-Gal) did not change the morphology of hepatocytes and decreased cell adhesion density to 70%–84% of that without the coating. However, the specific ammonia consumption rate of the culture on a TDOB-Gal-coated dish was 1.81 times that without coating, whereas those of TDOB-OH-and TDOB-Glc-coated dishes were 1.18 and 0.31 times that without coating, respectively. TDOB-Gal coating on a dish for adhesion culture decreased cell adhesion density only by 10% and did not disturb cell spreading. The coating of TDOB-Gal at 1/10 to 1/4 density of that for a monolayer resulted in a higher specific rate of ammonia consumption than that at monolayer density, and the rate reached 1.97 times that without coating. The ammonia consumption activity of hepatocytes was considered to be increased specifically not by the glucose residues but by the galactose residue of the glycolipid, independently of cell spreading.

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMRspectra in trifluoroethanol-H 2O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.

カルシトニンをモデルペプチドとして、それに糖鎖を付加することにより、糖鎖の構造や結合位置が、ペプチドの立体構造や生理活性に及ぼす影響を系統的に解析した。N-結合型糖鎖を付加した場合、立体構造には変化を与えなかったが、生理活性は主に糖鎖の構造によって変化することが明らかとなり、O-結合型糖鎖を付加した場合には、立体構造も生理活性も糖鎖の結合位置に依存した変化を受けることが明らかになった。糖鎖が、その構造や結合位置に依存して、ペプチドの立体構造や生理活性に影響を及ぼすことが系統的に示されたこれらの結果は、糖鎖を生理活性ペプチドの開発等に応用する際の指針になると考えられる。

Background. The human IgA1 hinge region is a unique mucin-like O-linked proline-rich glycopeptide, and its core peptide was found to be exposed aberrantly by the underglycosylation in IgA nephropathy (IgAN ). Introduction We describe here the presence of humoral immunity against the IgA1 hinge peptide epitope in IgAN and IgA nephropathy (IgAN ) is the most common glomerevaluate the relationship between the underglycosyl- ular disease which is characterized immunohistologation of the IgA1 hinge region and humoral immunity. ically by the predominant deposition of the IgA1 Method. The serum anti-IgA1 hinge peptide antibody subclass in the mesangial area [1,2]. The structural (anti-a1HP ab) titre was measured and compared characteristics of the IgA1 molecule recently were between the IgAN (n=37) and control groups (n=34) associated with the pathogenesis of IgAN, because the by enzyme-linked immunosorbent assay ( ELISA) IgA1 molecule is obviously diVerent from the other using a synthetic peptide corresponding to the human immunoglobulins in its hinge region which is a unique IgA1 hinge region, PVPSTPPTPSPSTPPTPSPS, as an mucin-like glycopeptide. The IgA1 hinge region has a antigen. Next, to evaluate the relationship between the proline-, serine- and threonine-rich amino acid underglycosylation of the IgA1 hinge region and the sequence in which the serine and threonine residues humoral immunity, the reactivity of the serum IgG are able to carry O-linked oligosaccharides consisting from the patients with IgAN against monoclonal IgA1 of sialic acid (NeuNAc), galactose (Gal ) and Nwhich had been digested enzymatically to remove the acetylgalactosamine (GalNAc) with microheterocarbohydrates from the IgA1 hinge region was meas- geneity [3,4]. Some investigators, including us, revealed ured by ELISA. the presence of defective O-glycans in the hinge region Results. The anti-a1HP ab titre was significantly higher of the IgA1 molecules derived from sera of patients in the IgAN group than in the control group (OD with IgAN [5‐9]. Additionally, we showed that the value: IgG class, 0.564±0.344 vs 0.331±0.154, P= core peptide of the IgA1 hinge region was exposed by 0.0014; IgM class, 0.272±0.148 vs 0.141±0.072, its underglycosylation in IgAN using a rabbit polyP

This study was performed to isolate and investigate the IgA1 that could accumulate in glomeruli (glomerulophilic IgA1). IgA1 was fractionated by the electric charge and the reactivity to Jacalin. Serum IgA1 of IgA nephropathy patients was separated and fractionated using a Jacalin column and subsequent ion-exchange chromatography. The fractions were divided into three groups of relatively cationic (C), neutral (N), and anionic (A). IgA1 was also divided into Jacalin low (L), intermediate (I), and high (H) affinity fractions by serial elu- tion using 25, 100, and 800 mM galactose. The left kidneys of Wistar rats were perfused with 2, 5, or 10 mg of each group of IgA1. The rats were sacrificed 15 min, 30 min, 3 h, or 24 h after the perfusion. The accumulation of each IgA1 in the glomeruli was then observed by immunofluorescence. The IgA1 of the fractions N and H separated by the two methods was definitely accumulated in the rat glomeruli with a similar pattern. The electrophoresis revealed that the macromolecular IgA1 was increased in fraction H compared with other frac- tions. Therefore, Jacalin high-affinity IgA1(fraction H) was applied on a diethylaminoethyl column and divided into elec- trically cationic (HC), neutral (HN), and anionic (HA). Only the asialo-Galb1,3GalNAc chain was identified in the fraction HN IgA1 by gas-phase hydrazinolysis. Furthermore, the IgA1 fraction was strongly recognized by peanut agglutinin, Vicia Villosa lectins, and antisynthetic hinge peptide antibody. These results indicated that the IgA1 molecules having the undergly- cosylated hinge glycopeptide played a certain role in the glo- merular accumulation of IgA1 in IgA nephropathy.

A part of human serum immunoglobulin A1(IgA1) was aggregated by treatment with neuraminidase. Aggregated IgA1 was separated from non-aggregated IgA1 by gel permeation chromatography. The prepared asialo-hinge glycopeptide (asialo-HGP) from both IgA1 subfractions was treated with β-galactosidase to determine the number of O -linked sugar chains attached on the hinge region. Removal of the galactose residue from asialo-HGP resulted in the HPLC separation of three major peaks. MALDI-TOFMS analysis of the glycopeptides also indicated the presence of three HGP components with three, four and five N -acetylgalactosamine (GalNAc) residues, respectively. Comparison of their relative content among the glycopeptide components showed a higher content of the HGP component with a lower number of GalNAc residues on aggregated IgA1. Thus, asialo-HGP prepared from aggregated IgA1 induced by neuraminidase treatment had an incomplete core structure of O -linked oligosaccharides. Especially, the result suggested that the reduced number of the attached O -linked oligosaccharides on IgA1 take part in phenomena such as self-aggregation of asialo-IgA1.

The aim of this study was to investigate the role of carbohydrate moieties attached to IgA 1 hinge region in IgA 1 self-aggregation and adhesion to extracellular matrix (ECM) proteins previously reported in IgA nephropathy. Serum IgAI samples isolated from healthy individuals were digested with neuraminidase (NA), NA + f3-galactosidase, and NA + 13-ga- lactosidase + a-N-acetylgalactosaminidase to remove the car- bohydrates from the hinge region and were named asialo, agalacto, and naked IgA I , respectively. First, polyacrylamide gel electrophoresis was performed under the native condition, and consequently, a broad band indicating IgA I self-aggrega- tion was clearly observed in asiabo, agalacto, and naked IgA I, but not in native IgA I. However, the broad band disappeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the nonreducing condition. Second, it was shown that IgAb adhesion activities to type IV collagen, fibronectin, and laminin were significantly higher in asiabo, agalacto, and naked IgAl than in native IgA1, using enzyme-linked immunosor- bent assay (asiabo, agalacto, and naked versus native: P < 0.0 1). In addition, agalacto IgA 1 had the highest affinity for all of the ECM proteins among the deglycosybated IgA I (agalacto versus asiabo and naked, P < 0.05). These results indicated that the removal of carbohydrates from the IgA I molecule resulted in noncovalent self-aggregation and a significant increase in adhesion to the ECM proteins. It was therefore suggested that the IgAl glycans played a protective role against aggregation and adhesion and that the underglycosylation of the IgA I molecule found in IgA nephropathy could be involved in the nonimmunobogic gbomerular accumulation of IgA I.

Glycopeptides containing the N-linked oligosaccharide from human serum IgA1 were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). Two glycopeptides, GP1 and GP2, prepared from the endoproteinase Asp-N digest of the IgA1 heavy chain, were derived from the CH2 domain (N-glycan site at Asn263) and the tailpiece portion (N-glycan site at Asn459), respectively. The structure of the attached sugar chain was deduced from the mass number of the glycopeptide and confirmed by a two-dimensional mapping technique for a pyridylaminated oligosaccharide. GP1 was composed of two major components having a fully galactosylated bianntena sugar chain with or without a bisecting N-acetylglucosamine (GlcNAc) residue. On the other hand, the GP2 fraction corresponded to the glycopeptides having a fully galactosylated and fucosylated bianntena sugar chain partly bearing a bisecting GlcNAc residue. Thus, the site-specific fucosylation of the N-linked oligosaccharide on the tailpiece of the α1 chain became evident for normal human serum IgA1.

Based on a plasmon response function in both amplitude and phase measured by a spectral interferometry combined with a near-field scanning optical microscopy (NSOM), the femtosecond plasmon pulse at gold nanostructures is deterministically tailored.

Spatiotemporal nanofocusing of surface plasmon polariton excited by femtosecond laser pulses on a sharp conical Au tip with a tip edge radius of few tens of nanometers is measured with cross-correlation dark-field microscopy.

Decaarginine-polyethylene glycol-conjugated 3,5-bis(dodecyloxy)benzamide/plasmid DNA [Arg10-polyethylene glycol (PEG)-lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated.R10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µm or 25 µm R10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light-electron microscopy (CLEM).EM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B-mediated gene delivery.We have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B-mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine-mediated carriers.

Coating of 3,4,5-tris(dodecyloxy)benzyl-β- d -galactopyranoside (TDOB-Gal) on a dish for suspension culture increased the specific ammonia consumption rate of primary rat hepatocytes to 2.4 times that in the case of rat hepatocytes cultured in a dish without coating while there was no increase in the specific urea production rate. TDOB-β- d -glucopyranoside (TDOB-Glc), -α- d -mannnoside, -β- d -mannoside, 2-acetamido-2-deoxy-β- d -glucopyranoside, and 2-acetamido-2-deoxy-β- d -galactopyranoside had almost no influence on the above-mentioned specific rates. In the ammonia loading assay, cells on the dish with TDOB-Gal and -Glc coatings produced glucose, suggesting gluconeogenesis.

Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering.

Previously, we have reported that decaarginine-conjugated PEG-lipids (R10B) efficiently delivered plasmid DNA (pDNA) into human cervical carcinoma HeLa cells via macropinocytosis; however, the mechanism of cellular uptake by R10B was not evaluated in other cell lines. In this study, we investigated the internalization mechanism by R10B/pDNA complex (R10B-lipoplex) in human prostate tumor PC-3 and human nasopharyngeal tumor KB cells, and compared with that in HeLa cells. Although it was necessary for R10B-lipoplex to associate with heparan sulfate (HS) on the cell surface in all cell lines, the R10B-lipoplex was internalized primarily through clathrin-mediated endocytosis in PC-3 and KB cells, and macropinosytosis in HeLa cells. In HeLa cells, treatment with the R10B-lipoplex induced the formation of lamellipodia for macropinocytosis, but did not in KB and PC-3 cells. Furthermore, the highest transfection efficiency by R10B-lipoplex was observed in HeLa cells. These findings indicated that the R10B-lipoplex induced the formation of lamellipodia in HeLa cells after binding to HS on the cells and was then internalized by macropinocytosis, which could induce high gene expression because of escaping degradation in lysosomes. Cell physiology might be a critical factor in cellular internalization and efficient transfection by cell penetration peptide.

3,4,5-Tris(octadecyloxy)benzyl alcohol, HO–Bzl(OC18)3, was prepared from gallic acid and stearyl bromide. Using conventional step-wise elongation, N,C-protected peptides, Fmoc–AAn–...–AA1–OBzl(OC18)3, were synthesized. The substituted benzyl esters were selectively cleaved by a treatment with 4 M hydrogen chloride in ethyl acetate to give Fmoc–AAn–...–AA1–OH and HO–Bzl(OC18)3. Thus, the substituted benzyl group is effective for the protection of C-terminal carboxyl groups in liquid-phase peptide synthesis. Because the substituted benzyl group has a moderately high molecular weight, Fmoc–AAn–...–AA1–OBzl(OC18)3 can be easily purified by size-exclusion chromatography; all protected peptides are eluted in the void fraction of a Sephadex LH-20 gel-filtration column. The combination of the carboxyl-protecting group Bzl(OC18)3 with simple purification by the gel-filtration gives a novel route for constructing combinatorial peptide libraries in the solution phase.

T-antigen (Galbeta1-3GalNAc-Thr/Ser) also known as Thomsen-Friedenreich (TF) antigen is the core 1 structure of O-linked mucin type glycans. In normal epithelium, the disaccharide structure is masked by terminal carbohydrate moieties, but is uncovered in most primary and metastatic epithelial malignant tumors. For the purpose of establishing cancer diagnosis and therapeutics, anti-T-antigen antibodies were isolated from a phage library displaying human single chain antibodies. A strategy similar to the previously published method (Sakai et al. Biochemistry. 2007; 46:253-262) was used to screen T-antigen specific antibodies, except that a different type of glycolipid was used for panning and screening. Eleven phage clones were isolated and characterized by DNA sequencing and ELISA, which revealed 4 groups of clones with T-antigen binding activity. One single chain antibody (scFv) protein, derived from phage clone 1G11, was expressed in Escherichia coli and purified to near homogeneity by two column chromatographies. ELISA and surface plasmon resonance analyses revealed that the purified 1G11 scFv bound to the T-antigen moiety of the neoglycolipid used. This study not only demonstrated the validity of our previously introduced strategy employing the phage display technology in constructing human scFvs against various carbohydrate antigens, but also provided us with various scFv genes that can lead to future development of antibody-based therapeutics.

Oligoarginine-conjugated lipids ((Arg)n-PEG-lipid) (n=4, 6, 8, and 10: number of arginine residues) are novel gene delivery vectors. We prepared two oligoarginine-modified liposomes using (Arg)n-lipid without and with poly(ethylene glycol) (PEG) spacer ((Arg)n-L and (Arg)n-PEG-L), and investigated the effect of PEG spacer and oligoarginine length of liposomes on cellular uptake, gene transfection, and its mechanism in HeLa cells, using complexes with plasmid DNA (DNA) or oligodeoxynucleotide. Transfection efficiency increased as the number of arginine residues increased and Arg10-PEG-L/DNA complexes (lipoplexes) showed the highest gene transfection efficiency among (Arg)n- and (Arg)n-PEG-lipoplexes. Arg4- and Arg4-PEG-lipoplexes were taken up greatly into cells, but showed lower transfection efficiency than Arg10- and Arg10-PEG-lipoplexes, respectively. The different gene expression by Arg4-L to Arg10-L with or without PEG spacer may be explained by the different intracellular uptake mechanism. The main cellular uptake mechanism of Arg10-L and Arg10-PEG-L was the macropinocytosis pathway, whereas that of Arg4-L and Arg4-PEG-L was not. PEG spacer was more effective for intracellular trafficking than Arg length and surface charge of lipoplex which depends on Arg length at the almost same size of lipoplexes. The findings suggested that Arg10-PEG-L was a superior vector since Arg10 induced the macropinocytosis uptake pathway.

As we have previously reported the delivery of plasmid DNA (DNA) complexed with oligoarginine-PEG artificial lipids (oligoarginine/DNA complexes), we focused on tetra- and decaarginine (Arg4, Arg10) to improve transfection efficiency by both the formation of oligoarginine-coated DNA complexed with protamine (PD), and the addition of Ca 2+ after formation of complexes. The efficiency of DNA condensation was determined by gel electrophoresis. Cellular uptake and transfection efficiency were evaluated in human cervical carcinoma HeLa cells using flow cytometry and luciferase assay. Oligoarginine-coated PD enhanced transfection efficiency significantly more than complexes where Arg10 in both vectors exhibited higher transfection efficiency than Arg4. As assessed by gel retardation assay, high gene expression by Arg10 may be explained by Arg4 binding DNA more strongly than Arg10. The addition of Ca 2+ to incubation medium increased transfection efficiency of Arg4-coated PD 70-fold, similar to that of Arg10-coated PD alone without an increase of cellular uptake, suggesting that Ca 2+ induced the release of DNA from complexes in endosomes. Only Arg4 with low cytotoxicity could gain an advantage from Ca 2+ in transfection, but Arg10 with relatively high cytotoxicity could not. The present results demonstrate that Arg4-coated PD with Ca 2+ has great potential as an efficient non-viral vector with low toxicity.

Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.

Camptothecin (CPT) has anticancer activity. While only the lactone form of CPT is biologically active, this form exhibits poor aqueous solubility. Pharmaceutical formulation of CPT incorporated in liposomes is of significant importance to develop the therapeutic utilization of CPT. The aim of this study was to increase incorporation efficiency and stability of CPT in liposomes composed of hydrogenated soybean phosphatidylcholine, cholesterol, and oleic acid (7 : 3 : 1, molar ratio), by incorporating three kinds of artificial lipids (DBs) (DB-liposome); 4-n-(M12B), 3,5-bis(B12B) and 3,4,5-tris(dodecyloxy)benzoic acid (T12B). The interaction of CPT with DB in the state of liposomes, was examined. In DB-liposomes presenting mean diameters of 150 nm, incorporation efficiency of CPT up to 55% and final drug to lipid molar ratio up to 0.07 were obtained when the liposomes were prepared at a feeding ratio of 1/30 (w/w) CPT/total lipid. However, in the optimal formulations, incorporated DB mol% was different; T12B and D12B were incorporated about one third and half mol% of M12B, respectively. Moreover, we demonstrated that T12B stabilized CPT in liposomes significantly compared with other DBs as measured by CPT release, and by steady state fluorescence polarization degree of CPT using intrinsic fluorescence of CPT. These findings suggested that in addition of contribution of phenyl group of DB, dodecyloxy group may interact strongly with lactone ring of CPT. The capacity to contain CPT interacted with DBs may be limited in liposomes. T12B may be incorporated in the interior of the bilayers, resulting in increase of incorporation stability of CPT. This finding demonstrates a potential application of the novel liposome formulation of CPT in drug delivery.

Camptothecin (CPT) is a strong antitumor agent, but its use limited by its low solubility and the instability of the active lactone form. To overcome these difficulties, liposomes incorporating CPT (CPT liposomes) were designed and tested. CPT liposomes were formulated by the addition of 3,5-bis(dodecyloxy)benzoic acid (DB) to polyethylene glycol-containing liposomes, and by coating the surface of the liposomes with human serum albumin (HSA, HSA-DB-L). HSA-DB-L successfully entrapped CPT with about 80% efficiency and with a particle size of about 150 nm. HSA-DB-L showed attenuated drug release and storage stability. Pharmacokinetics studies in mice showed that i.v. injection of HSA-DB-L (2.5 mg/kg) led to prolonged circulation in the plasma; the area under the curve was 22-fold higher than that of CPT solution. The tumor growth in mice with subcutaneous transplantation of colon 26 tumor cells was significantly inhibited after a single i.v. injection of HSA-DB-L at a dose of 15 mg/kg without any significant body weight loss. HSA-DB-L increased the accumulation of CPT in tumor tissue significantly (9.6-fold) more efficiently than CPT solution 24 h after i.v. injection. These findings suggest that HSA-DB-L could increase the stability and the antitumor effect of CPT. CPT delivery by novel liposome formulations is a potential approach for effective treatment of cancer.

Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyses the hydrolysis and transglycosylation of asparagine-linked oligosaccharides of glycoproteins with retention of anomeric configuration. Glu-173 of Endo-A is a catalytically essential amino acid residue, and the corresponding residue is conserved in all GH family 85 members. The catalytic activity of Endo-A E173A mutant was rescued by the addition of sodium azide or sodium formate. Furthermore, the produced beta-glycosyl azide (Man(5)GlcNAc-beta-N(3)) retained the anomeric configuration, indicating that Glu-173 is the catalytic acid-base residue of Endo-A. This is the first identification of the catalytic residue for GH family 85 endo-beta-N-acetylglucosaminidases.

Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.

The intracellular delivery of proteins using cell-penetrating peptides (CPPs) including oligoarginine (oligo-Arg) carriers raises the possibility of establishing novel therapeutic methods. We compared the effect of the length of oligo-Arg in modified liposomes ((Arg)(n)-L; n = 4, 6, 8, 10) on the delivery of proteins by flow cytometry, fluorescence microscopy, and spectrofluorimetry. As a free liposome, Arg4-modified liposome Arg4-L was most efficiently internalized in cells. The efficiency decreased depending on the length of oligo-Arg. For the intracellular delivery of proteins, (Arg)(n)-L was physically associated with proteins. Concerning the effect of oligo-Arg length, liposome/protein complexes showed a different behavior. Arg4-L carried bovine serum albumin (BSA, 66 kDa) and beta-galactosidase (beta-Gal, 120 kDa) 6-fold higher than free BSA and free beta-Gal. Arg10-L showed similar performance for these two proteins to Arg4-L. The enzymatic activity of beta-Gal in the cells showed that proteins were transported as a biologically active form. Arg10-L carried 100-fold more immunoglobulin G (IgG, 150 kDa) than free IgG, and 3-fold more than Arg4-L into cells. Shorter oligo-Arg chain on liposomes may be enough for liposomes alone to be taken up in cells, but more Arg residues may be needed to form a complex with high molecular weight proteins and deliver them into cells. This information will aid in the design of (Arg)(n)-L as a carrier for delivering proteins into cells.

text/plain, application/pdf, 学術雑誌論文, How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to α-Man, Neu5Acα2,6Galβ1,4Glc, and α-Gal sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing α-Man or α-NeuAc but not to those possessing β-Gal or β-GlcNAc residue. ELISA showed that trypsin binds to six kinds of biotinylated glycoproteins possessing high mannose-type and complex type N-glycans but not to bovine submaxillary mucin, which possesses only O-glycans. Further, the binding of trypsin to glycoproteins was differentially changed by treatments with sequential exoglycosidases, endoglycosidase H, or N-glycosidase F. Quantitative kinetic studies indicated that PMSF-treated trypsin binds with bovine thyroglobulin with the affinity constant of 1010 M-1, which was the highest among the glycoproteins examined, and that α-galactosidase treatment decreased it to 105 M-1. PMSF-treated trypsin bound to other glycoproteins inc\luding ovomucoid, a trypsin inhibitor, with the affinity constants of 108-105 mol-1 and were markedly changed by glycosidase treatments in manners consistent with the sugar-binding specificities suggested by ELISA. Thus, the binding site for glycans was shown to be distinct from the catalytic site, allowing trypsin to function as an uncompetitive activator in the hydrolysis of a synthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment with PMSF or soybean trypsin inhibitor. The results indicate the presence of an allosteric regulatory site on trypsin that sugar-specifically interacts with glycoproteins in addition to the proteolytic catalytic site.

The design, synthesis, and evaluation of in vitro gene delivery efficacy of a novel series of oligo-Arg-lipid conjugates are described. 3,5-Bis(dodecyloxy)benzamide (BDB) was employed as the lipid component, and a poly(ethylene glycol) (PEG) spacer was introduced between the C-terminal of oligo-Arg and the amide group of BDB. Four derivatives with various oligo-Arg lengths (ArgN-PEG-BDB; N = 4, 6, 8, 10: the number of arginine residues) were prepared, and the effect of oligo-Arg length on the gene transfection was investigated in HeLa cells. Transfection efficiency increased as the number of arginine residues increased. Arg10-PEG-BDB showed the highest transfection efficiency, without severe toxicity to cells. These findings well corresponded to the cellular association of the Arg-PEG-BDB/DNA complex determined by flow cytometry. Even in the presence of serum, Arg10-PEG-BDB achieved appreciable cellular association and attained high gene expression. Thus, Arg10-PEG-BDB is potentially a simple and useful gene delivery tool, because one need only to mix it with plasmid DNA and apply the complexes to the cells even in a serum-containing medium.

Previous studies have shown that different calcitonins interact with planar lipid membranes to form ion channels. In this study, glycosylation of eel calcitonin (eCt) at different positions (Ct3-GlcNAc, Ct14-GlcNAc, Ct20-GlcNAc, Ct26-GlcNAc) is shown to preserve molecular structure and slightly change the energy of incorporation and channel formation in planar lipid bilayers made up of palmitoyl-oleoyl-phosphatidylcholine:dioleoyl phosphatidyl-glycerol (85:15, w:w). The voltage needed to form channels decreased as the attached carbohydrate moved toward the C-terminal (eCt = Ct3-GlcNAc > Ct14-GlcNAc = Ct20-GlcNAc > Ct26-GlcNAc). Interestingly, all the Cts tested maintain the characteristic voltage–conductance dependence found for other Cts, the only channel properties modified concern ion selectivity, that shift toward anion selectivity (eCt = 0.97, Ct3-GlcNAc = 0.49, Ct14-GlcNAc = 0.41, Ct20-GlcNAc = 0.36, Ct26-GlcNAc = 0.47). These aspects would be useful in managing peptide properties for biotechnological and therapeutic applications considering the physiological nature of this peptide.

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.