Yoshinao Wada

Site-specific analysis of N-glycans on haptoglobin in sera of patients with pancreatic cancer: a novel approach for the development of tumor markers.

It was found in our previous studies that the concentration of fucosylated haptoglobin had increased in the sera of patients with pancreatic cancer (PC) compared to those of other types of cancer and normal controls. Haptoglobin, an acute phase protein, has four potential N‐glycosylation sites, although it remains unknown which site is responsible for the change in fucosylated N‐glycans. In the present study, site‐specific N‐glycan structures of haptoglobin in sera obtained from patients with PC or chronic pancreatitis (CP) were analyzed using liquid chromatography‐electrospray ionization mass spectrometry. Mass spectrometry analyses demonstrated that concentrations of total fucosylated di‐, tri‐ and tetra‐branched glycans of haptoglobin increased in the sera of PC patients. Tri‐antennary N‐glycans containing a Lewis X‐type fucose markedly increased at the Asn211 site of haptoglobin N‐glycans. While fucosylated N‐glycans derived from serum haptoglobin of patients with CP slightly increased, di‐fucosylated tetra‐antennary N‐glycans were observed only at this site in PC patients, and were absent in the haptoglobin of normal controls and individuals with CP. Thus, the present study provides evidence that site‐specific analyses of N‐glycans may be useful as novel tumor markers for PC.

Comparison of Methods for Profiling O-Glycosylation HUMAN PROTEOME ORGANISATION HUMAN DISEASE GLYCOMICS/PROTEOME INITIATIVE MULTI-INSTITUTIONAL STUDY OF IgA1

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiatives activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

N-Glycosylation of the β-Propeller Domain of the Integrin α5 Subunit Is Essential for α5β1 Heterodimerization, Expression on the Cell Surface, and Its Biological Function

The N-glycosylation of integrin α5β1 is thought to play crucial roles in cell spreading, cell migration, ligand binding, and dimer formation, but the underlying mechanism remains unclear. To investigate the importance of the N-glycans of this integrin in detail, sequential site-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on theα5 integrin. Removal of the putative N-glycosylation sites on the β-propeller, Thigh, Calf-1, or Calf-2 domains of the α5 subunit resulted in a decrease in molecular weight compared with the wild type, suggesting that all of these domains contain attached N-glycans. Importantly, the absence of N-glycosylation sites (sites 1–5) on the β-propeller resulted in the persistent association of integrin subunit with calnexin in the endoplasmic reticulum, which subsequently blocked heterodimerization and its expression on the cell surface. Interestingly, the activities for cell spreading and migration for the α5 subunit carrying only three potential N-glycosylation sites (3–5 sites) on theβ-propeller were comparable with those of the wild type. In contrast, mutation of these three sites resulted in a significant decrease in cell spreading as well as functional expression, although the total expression level of the Δ3–5 mutant on the cell surface was comparable with that of wild type. Furthermore, we found that site 5 is a most important site for its expression on the cell surface, whereas the S5 mutant did not show any biological functions. Taken together, this study reveals for the first time that the N-glycosylation on the β-propeller domain of the α5 subunit is essential for heterodimerization and biological functions of α5β1 integrin and might also be useful for studies of the molecular structure.

Quantitation of saccharide compositions of O-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis.

Profiling of oligosaccharide structures is widely utilized for both identification and evaluation of glycobiomarkers, and site-specific profiling of N-linked glycans of glycoproteins is conducted by mass spectrometry of glycopeptides. However, our knowledge of mucin-type O-glycans including site occupancy and profile variance, as well as attachment sites, is quite limited. Saccharide compositions and site-occupancy of O-glycans were calculated from the signal intensity of glycopeptide ions in the mass spectra and tandem mass spectra from electron transfer dissociation. The results for two major plasma glycoproteins, IgA1 and hemopexin, representing clustered and scattered O-glycan attachments, respectively, indicated that the variability in modifications among individuals is so small as to justify rigorous standards enabling reliable detection of disease-related alterations. Indeed, this method revealed a novel abnormality associated with rheumatoid arthritis: a significant decrease in the N-acetylgalactosamine content of IgA1 O-glycans, indicating that the glycosylation abnormality is not limited to hypogalactosylation of IgG N-glycans in chronic inflammatory conditions.

Site-directed mutagenesis in haemoglobin: Functional role of tyrosine-42(C7)α at the α1-β2 interface☆

Abstract To clarify the functional role of Tyr-42(C7)α, which forms a hydrogen bond with Asp-99(G1)β at the α1-β2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42α was replaced by Phe (Hb Phe-42α) or His (Hb His-42α), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99β mutants, Hb Phe-42α showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42α, the deoxy T quaternary structure is highly destabilized and the strain imposed on the FeN e (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42α, Hb His-42α showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42α and Asp-99β. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42α and Asp-99β plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.

Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

Calponin 3 (CNN3), an actin binding molecule, negatively regulates trophoblast differentiation and fusion. CNN3 phosphorylation modulates the actin-binding capacity of CNN3 and most probably regulates cytoskeleton remodeling that renders cells capable of undergoing fusion.

Molecular Cloning and Characterization of a Human β-Gal-3′-sulfotransferase That Acts on Both Type 1 and Type 2 (Galβ1–3/1–4GlcNAc-R) Oligosaccharides

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3′-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864–4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5′- and 3′-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing β-galactosides such asN-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Galβ1–3GalNAcα-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO3-3Galβ1–3GlcNAcβ1–3Galβ1–4Glc-PA by two-dimensional 1H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Galβ1–3GlcNAc-R) and type 2 (Galβ1–4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel β-Gal-3′-sulfotransferase gene family.

N-Glycosylation of Laminin-332 Regulates Its Biological Functions A NOVEL FUNCTION OF THE BISECTING GlcNAc

Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of β1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and β1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired α3β1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.

Role of Neutrophils in Matrix Metalloproteinase Activity in the Preimplantation Mouse Uterus

Abstract Matrix metalloproteinases (MMPs) have been implicated in embryonal implantation processes such as trophoblast invasion and decidualization. The temporal and spatial distributions of MMP bioactivities were analyzed by in situ zymography, which indicated these activities to be markedly increased in the postcoital mouse uterus compared with those in the later implantation stage. Activity was ascribed to proMMP9, which moved from the uterine serosa to the endometrium but was not associated with mRNA up-regulation. The activity was colocalized with infiltrating neutrophils, and neutropenic mice did not exhibit MMP9 expression. Removing the seminal vesicles from male mice abolished the postcoital increase in MMP9 in the female. These results indicate the major MMP activity in the preimplantation uterus to originate in proMMP9-bearing neutrophils attracted by seminal plasma. Considering our results together with those of previous reports of reduced fertility in Mmp9-deficient female mice, we speculate that neutrophil infiltration participates in the extracellular matrix degradation needed to support pregnancy.

Advanced analytical methods for hemoglobin variants.

Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes.