Daisuke Takakura

β-Galactoside α2,6-Sialyltranferase 1 Promotes Transforming Growth Factor-β-mediated Epithelial-Mesenchymal Transition

Background: Molecular mechanisms underlying the effect of sialylation on tumor progression remain unclear. Results: ST6GAL1 promoted the TGF-β-induced EMT through down-regulation of E-cadherin-mediated cell adhesion and up-regulation of integrin-mediated cell migration. Conclusion: Expression of ST6GAL1 is critical for sufficient induction of EMT. Significance: α2,6-Sialylation of N-glycans may play a role in EMT. β-Galactoside α2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal α2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and α2,6-sialylated N-glycans are up-regulated during TGF-β-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-β-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-β-induced EMT. Overexpressing β-galactoside α2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for α2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-β-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.

Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy.

An oncogenic protein Golgi phosphoprotein 3 up-regulates cell migration via sialylation

Background: Molecular mechanisms of the effect of the GOLPH3 oncogenic protein on tumorigenesis remain unclear. Results: GOLPH3 specifically up-regulates sialylation of integrin N-glycans, promotes sialylation-dependent cell migration, and affects AKT signaling. Conclusion: GOLPH3 affects cell biological functions through a specific regulation of sialylation. Significance: The sialylation of N-glycans is important for functions of GOLPH3. Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.

Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

Background: 20 read-through mutations that produce C-terminally extended proteins are related to human hereditary disorders. Results: The C-terminal extended proteins of mouse cFLIP-L (cellular FLICE-like apoptosisinhibitory protein) and human PNPO (pyridoxamine 5-phosphate oxidase) and HSD3B2 (3-hydroxysteroid dehydrogenase type II) are ubiquitylated and degraded, involving an E3 ligase, TRIM21, for cFLIP-L and PNPO degradation. Conclusion: Read-through mutant cFLIP-L, PNPO, and HSD3B2 are degraded by the ubiquitin-proteasome system. Significance: Degradation of read-through mutant proteins may cause hereditary disorders. During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

Selective glycopeptide profiling by acetone enrichment and LC/MS

UNLABELLED LC/MS is commonly used for site-specific glycosylation analysis of glycoproteins in cells and tissues. A limitation of this technique is the difficulty in acquiring reliable mass spectra for glycopeptides, mainly due to their high heterogeneity and poor hydrophobicity. Here, we establish a versatile method for efficient glycopeptide enrichment to acquire reliable mass spectra. Several lines of evidence using model glycoproteins suggest that our method is based on the different solubility between non-glycosylated and glycosylated peptides in acetone. We also provide data showing that the acetone-precipitated glycopeptide enrichment was successful in acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide in crude human serum. We propose that this method is a powerful tool for the acquisition of reliable mass spectra from trace amounts of glycopeptides and an alternative to lectin affinity enrichment. BIOLOGICAL SIGNIFICANCE In this study, we established a versatile method for glycopeptide enrichment to acquire reliable mass spectra because of the limitation of conventional enrichment methods, such as lectin affinity chromatography. Our enrichment method is capable of isolating glycopeptides from complex peptide mixtures such as crude serum.

Change in N-Glycosylation of Plasma Proteins in Japanese Semisupercentenarians

An N-glycomic analysis of plasma proteins was performed in Japanese semisupercentenarians (SSCs) (mean 106.7 years), aged controls (mean 71.6 years), and young controls (mean 30.2 years) by liquid chromatography/mass spectrometry (LC/MS) using a graphitized carbon column. Characteristic N-glycans in SSCs were discriminated using a multivariate analysis; orthogonal projections to latent structures (O-PLS). The results obtained showed that multi-branched and highly sialylated N-glycans as well as agalacto- and/or bisecting N-glycans were increased in SSCs, while biantennary N-glycans were decreased. Since multi-branched and highly sialylated N-glycans have been implicated in anti-inflammatory activities, these changes may play a role in the enhanced chronic inflammation observed in SSCs. The levels of inflammatory proteins, such as CRP, adiponectin, IL-6, and TNF-α, were elevated in SSCs. These results suggested that responses to inflammation may play an important role in extreme longevity and healthy aging in humans. This is the first study to show that the N-glycans of plasma proteins were associated with extreme longevity and healthy aging in humans.

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Characterization of N‐glycan heterogeneities of erythropoietin products by liquid chromatography/mass spectrometry and multivariate analysis

RATIONALE Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes. METHODS The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs. RESULTS Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data. CONCLUSIONS PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products.

An improved in‐gel digestion method for efficient identification of protein and glycosylation analysis of glycoproteins using guanidine hydrochloride

In‐gel digestion followed by LC/MS/MS is widely used for the identification of trace amounts of proteins and for the site‐specific glycosylation analysis of glycoproteins in cells and tissues. A major limitation of this technique is the difficulty in acquiring reliable mass spectra for peptides present in minute quantities and glycopeptides with high heterogeneity and poor hydrophobicity. It is considered that the SDS used in electrophoresis can interact with proteins noncovalently and impede the ionization of peptides/glycopeptides. In this study, we report an improved in‐gel digestion method to acquire reliable mass spectra of a trace amount of peptides/glycopeptides. A key innovation of our improved method is the use of guanidine hydrochloride, which forms complexes with the residual SDS molecules in the sample. The precipitation and removal of SDS by addition of the guanidine hydrochloride was successful in improving the S/N of peptides/glycopeptides in mass spectra and acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide.