Pros and cons of using aberrant glycosylation as companion biomarkers for therapeutics in cancer

( Jeong Gu Kang ),( Jeong Heon Ko ),( Yong Sam Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.12

Cancer treatment has been stratified by companion biomarker tests that serve to provide information on the genetic status of cancer patients and to identify patients who can be expected to respond to a given treatment. This stratification guarantees better efficiency and safety during treatment. Cancer patients, however, marginally benefit from the current companion biomarker-aided treatment regimens, presumably because companion biomarker tests are dependent solely on the mutation status of several genes status quo. In the true sense of the term, "personalized medicine", cancer patients are deemed to be identified individually by their molecular signatures, which are not necessarily confined to genetic mutations. Glycosylation is tremendously dynamic and shows alterations in cancer. Evidence is accumulating that aberrant glycosylation contributes to the development and progression of cancer, holding the promise for use of glycosylation status as a companion biomarker in cancer treatment. There are, however, several challenges derived from the lack of a reliable detection system for aberrant glycosylation, and a limited library of aberrant glycosylation. The challenges should be addressed if glycosylation status is to be used as a companion biomarker in cancer treatment and contribute to the fulfillment of personalized medicine. [BMB reports 2011; 44(12): 765-771]

Specific Biological Activity of Equine Chorionic Gonadotropin (eCG) Glycosylation Sites in Cells Expressing Equine Luteinizing Hormone/CG (eLH/ CG) Receptor

Munkhzaya Byambaragchaa,Seung-Hee Choi,Hyo-Eun Joo,Sang-Gwon Kim,Yean-Ji Kim,Gyeong-Eun Park,Myung-Hwa Kang,민관식 한국발생생물학회 2021 발생과 생식 Vol.25 No.4

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)- like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit56 N-linked glycosylation site; eCGβ-D/ α, deletion of the O-linked glycosylation sites at the β-subunit, and eCGβ-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC50 levels of eCGβ/αΔ56, eCGβ-D/ α, and eCGβ-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wildtype eCG (141.9 nmol/104 cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/ CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of receCG glycosylation sites in equidaes.

In Vitro Glycosylation of Peptide (RKDVY) and RNase A by PNGase F

( Su Jin Park ),( Ji Youn Lee ),( Tai Hyun Park ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.2

모체의 당뇨병이 흰 쥐 태자 폐세포의 glycosylation에 미치는 영향

홍혜남,김동호 울산대학교 의과대학 1992 울산의대학술지 Vol.1 No.1

Stretozotocin으로 유발된 모체의 당뇨병이 태자 폐세포의 당질화(glycosylation) 과정에 미치는 영향을 알아보기 위하여 태자기 19일과 21일째의 폐조직을 ConA, WGA, UEAL, RCAL, MPA등의 lectin으로 염색하여 결합양상을 조사하였으며, image analyzer를 사용하여 형태학적인 계측을 시행하여 얻은 결과는 아래와 같다. 태자기 21일째의 폐조직에서 bronchiolar epithelium이 차지하는 면적은 두 군데에서 비슷하였으나 alveolar epithelial cell과 mesenchymal cell이 차지하는 면적의 비율은 실험군이 74.2%, 정상군이 56.8%를 나타내었으며, air space의 면적비율은 실험군에서 낮게 나타났다. 다섯 종류의 lectin으로 조직화학염색을 시행한 결과 α-mannose, N-acetylgucosamine, fucose, β-galactose와 각각 결합하는 ConA, WGA, UEAI, RCAI의 binding affinities는 정상군과 큰 차이를 나타내지 않았으나 N-acetyl-D-galactosamine과 결합하는 MPA의 binding intensity는 실험군에서 감소되어 나타났다. 이는 stretozotocin으로 유발된 모체의 당뇨병이 태자 폐조직의 O-linked glycosylation과정 중 어느 단계를 저해시켜 나타난 결과가 아닌가 추측된다. This study was to investigate the effects of maternal diabetes on glycosylation in the lung of the fetal rat using lectin histochmistry. Maternal diabetes was induced by intraperitoneal injection of stretozotocin(75mg/kg the body weight) into pregnant Sprague-Dawley rats on the 7th day of geastation. Fetuses of streptozocin-induced diabetic rats exhibited delayed lung maturation and reduced air space. In lectin histochemistry, the binding of Maclura pomifera(MPA) to fetal lings from diabetic mothers was reduced, but no significant changes in the binding of Concanavalin A(ConA). Wheat germ agglutinin(WGA), Ricinus communis I (RSAI) and Ulex eupopaeus I (UEAI) were noted. Because the MPA has affinity to terminal N-acetyl-D-galactosamine residues con stantly linked O-glycosidically to serine or threonine, the present finding may indicate that maternal diabetes interfere with the processes of O-linked glycosylation in fetal rat lung.

A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

Morokuma, Daisuke,Hino, Masato,Tsuchioka, Miho,Masuda, Akitsu,Mon, Hiroaki,Fujiyama, Kazuhito,Kajiura, Hiroyuki,Kusakabe, Takahiro,Lee, Jae Man Korean Society of Sericultural Science 2018 International Journal of Industrial Entomology Vol.36 No.1

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.

N-glycosylation in desmoglein is required for desmosome assembly

( Seon Pil Jin ),( Jang Hee Oh ),( Eun Young Seo ),( Jin Ho Chung ) 대한피부과학회 2015 대한피부과학회 학술발표대회집 Vol.67 No.2

Background: Desmogleins are considered to have an N-linked glycosylation. The function of N-glycosylation in desmogleins has only been investigated in terms of antigenicity. However, the effect of N-glycosylation onmediating cell adhesion, the major role of desmogleins in a physiologic state, is to be elucidated. Results: When tunicamycin was treated to the cultured keratinocyte, western blotting showed shifted band of desmogleins, which means the reduction in size and deglycosylation of the target protein. The quantity of desmogleins also decreased. And to see if the cell adhesion strength is reduced at this deglycosylated state, dissociation assay was performed. The number of cell fragments revealed that tunicamycin treatment dissociated cell sheets 2.6 fold more than control. Decreased amount of desmogleins cannot fully explicate the disruption of adhesion, seeing that the result of dissociation assay comparing the treatment of tunicamycin to that of salubrinal which attenuates protein translation. Immunocytofluorescence showed abberant feature of desmogleins, but seemed to appear normal trafficking to the cell membrane. And under the transmission electron microscope, the outer and inner dense plaque became thinner in tunicamycin treatment than control. Thestability of desmogleins also significantly decreased in tunicamycin treated cells. Conclusion: Taken together, inhibition of N-glycosylation disrupts assembly of desmsomes by producing abnormal desmoglein.

알부민과 Warfarin의 결합에 미치는 Glycosylation의 효과

배진우 건국대학교 의과학연구소 1991 건국의과학학술지 Vol.1 No.-

One of the most important functions of albumin is carrier for exogenous substances, especially drugs and endogenous substances such as hormones and waste products arising from metabolism. Glycosylated albumin is made from sterile incubation of albumin with glucose. After incubation of warfarin in glycosylated albumin, the free warfarin is separated from albumin bound warfarin with equilibrium dialysis and ultrafiltration. The amount of free warfarin equilibrium dialysis is slightly increased than that from ultrafiltration. But the amount of the free warfarin from variable grade of glycosylation is not different. Therefore the glycosylation of albumin has no influence on the binding of warfarin with human-serum-albumin.

A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

( Daisuke Morokuma ),( Masato Hino ),( Miho Tsuchioka ),( Akitsu Masuda ),( Hiroaki Mon ),( Kazuhito Fujiyama ),( Hiroyuki Kajiura ),( Takahiro Kusakabe ),( Jae Man Lee ) 한국잠사학회 2018 International Journal of Industrial Entomology Vol.36 No.1

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.

Exploring the Nucleophilic N- and S-Glycosylation Capacity of Bacillus licheniformis YjiC Enzyme

( Puspalata Bashyal ),( Samir Bahadur Thapa ),( Tae-su Kim ),( Ramesh Prasad Pandey ),( Jae Kyung Sohng ) 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.7

YjiC, a glycosyltransferase from Bacillus licheniformis, is a well-known versatile enzyme for glycosylation of diverse substrates. Although a number of O-glycosylated products have been produced using YjiC, no report has been updated for nucleophilic N-, S-, and C- glycosylation. Here, we report the additional functional capacity of YjiC for nucleophilic N- and S- glycosylation using a broad substrate spectrum including UDP-α-D-glucose, UDP-N-acetyl glucosamine, UDP-N-acetyl-galactosamine, UDP-α-D-glucuronic acid, TDP-α-L-rhamnose, TDP-α-D-viosamine, and GDP-α-Lfucose as donor and various amine and thiol groups containing natural products as acceptor substrates. The results revealed YjiC as a promiscuous enzyme for conjugating diverse sugars at amine and thiol functional groups of small molecules applicable for generating glycofunctionalized chemical diversity libraries. The glycosylated products were analyzed using HPLC and LC/MS and compared with previous reports.

In-vivo Glycosylation of genistein in E. coli using GmIF7GT from Glycine max

Niranjan Koirala,Ramesh Prasad Pandey,Na-Rae Lee,Jae Kyung Sohng 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1

Isoflavonoids, polyphenols are biologically active natural products which are abundantly present in soyabean seeds during development. Clinical studies have suggested positive effects of isoflavonoids in human health and nutrition, such as reduction in the risks of hormonally dependent cancers, menopausal symptoms, osteoporosis, and cardiovascular disease. However, the insolubility and instability of those polyphenols prevent the wide applications of these medicinally important compounds. Therefore, the generation of isoflavonoid derivatives by glycosylation gained much attention and interest. In this study, we used E.coli BL21 (DE3) as an expression host for gmIF7Gt, a Glycine max derived glycosyl transferase, cell biocatalyst for the production of glycosylated derivatives of genistein. The supplementation of 0.2mM of genistein in the growing induced culture of E. coli BL21 (DE3) harboring PET15b-GmIF7GT resulted novel spots in TLC. These spots were further analyzed by HPLC and LC-TOF ESI/MS. The exact molecular mass analysis confirmed the production of Genistein glucoside. Further, structural elucidation and product enhancement is necessary for large scale production of this compound. However, this study reveals a method that might be useful for the biosynthesis of glycosylated isoflavonoids and related compounds by in-vivo glycosylation.