Site-Specific Introduction of Sialic Acid into Insulin

2004 ◽  
Vol 116 (12) ◽  
pp. 1542-1546 ◽  
Author(s):  
Masaaki Sato ◽  
Reiko Sadamoto ◽  
Kenichi Niikura ◽  
Kenji Monde ◽  
Hirosato Kondo ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Site Specific

scholarly journals Cross-species comparison of site-specific evolutionary-rate variation in influenza haemagglutinin

2013 ◽  
Vol 368 (1614) ◽  
pp. 20120334 ◽  
Author(s):  
Austin G. Meyer ◽  
Eric T. Dawson ◽  
Claus O. Wilke
Keyword(s):  
Protein Structure ◽  
Sialic Acid ◽  
Avian Influenza ◽  
Evolutionary Rate ◽  
Solvent Accessibility ◽  
Rate Variation ◽  
Structural Constraints ◽  
Binding Region ◽  
Site Specific ◽  
Sialic Acid Binding

We investigate the causes of site-specific evolutionary-rate variation in influenza haemagglutinin (HA) between human and avian influenza, for subtypes H1, H3, and H5. By calculating the evolutionary-rate ratio, ω = d N /d S as a function of a residue's solvent accessibility in the three-dimensional protein structure, we show that solvent accessibility has a significant but relatively modest effect on site-specific rate variation. By comparing rates within HA subtypes among host species, we derive an upper limit to the amount of variation that can be explained by structural constraints of any kind. Protein structure explains only 20–40% of the variation in ω . Finally, by comparing ω at sites near the sialic-acid-binding region to ω at other sites, we show that ω near the sialic-acid-binding region is significantly elevated in both human and avian influenza, with the exception of avian H5. We conclude that protein structure, HA subtype, and host biology all impose distinct selection pressures on sites in influenza HA.

scholarly journals Post-Glycosylation Modification of Sialic Acid and Its Role in Virus Pathogenesis

Vaccines ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 171 ◽  
Author(s):  
Park
Keyword(s):  
Sialic Acid ◽  
Mammalian Cells ◽  
Biological Significance ◽  
Extraction Methods ◽  
Sialic Acids ◽  
Neuraminic Acid ◽  
Host Cells ◽  
Viral Receptor ◽  
Site Specific ◽  
Viral Binding

Sialic acids are a family of nine carbon keto-aldononulosonic acids presented at the terminal ends of glycans on cellular membranes. α-Linked sialoglycoconjugates often undergo post-glycosylation modifications, among which O-acetylation of N-acetyl neuraminic acid (Neu5Ac) is the most common in mammalian cells. Isoforms of sialic acid are critical determinants of virus pathogenesis. To date, the focus of viral receptor-mediated attachment has been on Neu5Ac. O-Acetylated Neu5Acs have been largely ignored as receptor determinants of virus pathogenesis, although it is ubiquitous across species. Significantly, the array of structures resulting from site-specific O-acetylation by sialic acid O-acetyltransferases (SOATs) provides a means to examine specificity of viral binding to host cells. Specifically, C4 O-acetylated Neu5Ac can influence virus pathogenicity. However, the biological implications of only O-acetylated Neu5Ac at C7–9 have been explored extensively. This review will highlight the biological significance, extraction methods, and synthetic modifications of C4 O-acetylated Neu5Ac that may provide value in therapeutic developments and targets to prevent virus related diseases.

scholarly journals A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates

2021 ◽  
Vol 9 ◽  
Author(s):  
Qiong Wang ◽  
Yan Wang ◽  
Shuang Yang ◽  
Changyi Lin ◽  
Lateef Aliyu ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Viral Envelope ◽  
Site Specific ◽  
S Protein ◽  
Hek Cells ◽  
High Mannose ◽  
The Impact ◽  
S Proteins ◽  
Spike Proteins

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

Fabrication of β-cyclodextrin and sialic acid copolymer by single pot reaction to site specific drug delivery

2020 ◽  
Vol 13 (1) ◽  
pp. 1397-1405 ◽  
Author(s):  
Parbeen Singh ◽  
Xiaohong Ren ◽  
Yaping He ◽  
Li Wu ◽  
Caifen Wang ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Sialic Acid ◽  
Specific Drug ◽  
Site Specific ◽  
Acid Copolymer

THE SITE-SPECIFIC PROTEOLYSIS OF ESCHERICHIA COLI SIALIC ACID SYNTHASE AT LYS280

2002 ◽  
Author(s):  
Chun-Hung Lin ◽  
Tzann-Shun Hwang ◽  
Chih-Hung Hung ◽  
Chin-Fen Teo
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Site Specific

scholarly journals Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants

2007 ◽  
Vol 282 (49) ◽  
pp. 35491-35501 ◽  
Author(s):  
Ofer Cohen ◽  
Chanoch Kronman ◽  
Arie Lazar ◽  
Baruch Velan ◽  
Avigdor Shafferman
Keyword(s):  
Polyethylene Glycol ◽  
Sialic Acid ◽  
Chemical Modification ◽  
Inverse Relationship ◽  
Direct Relationship ◽  
Rank Order ◽  
Asialoglycoprotein Receptor ◽  
Protein Surface ◽  
Site Specific

Cholinesterases are efficient scavengers of organophosphates and are currently being developed as drugs for treatment against poisoning by such compounds. Recombinant ChE bioscavengers have very short circular longevity, a limitation that can be overcome by complex post-translation manipulations or by chemical modification such as polyethylene glycol conjugation. Series of multiple Lys-Ala mutants of human acetylcholinesterase were prepared allowing the generation of homogenous and well defined polyethylene-glycol conjugated AChEs with either one, two, three, four, or five appended polyethylene glycol (PEG) moieties/molecule. The rank order of circulatory longevity of these molecules was dependent on the number of PEG appendages up to a certain threshold: 5 = 4 > 3 > 2 > 1 > 0. Hypolysine acetylcholinesterases (AChEs) carrying the same number of PEGs, and therefore with identical masses, allowed us to demonstrate that circulatory longevity correlates with the predicted extent of concealment of the AChE surface. Furthermore, circulatory profiles of high number and low number PEG-AChEs differing in their sialic acid contents demonstrate a direct relationship between PEG loading and the effective seclusion of AChE from the hepatic asialoglycoprotein receptor clearance system. Finally, an inverse relationship is found between the extent of PEG loading and the ability of the human acetylcholinesterase to elicit specific anti-HuAChE antibodies. In conclusion, these findings suggest that for the extension of circulatory longevity, protein surface domain concealment exerted by polyethylene glycol attachment is at least as important as its effect on size enlargement and highlights the role of PEG attachment in masking interactions between biomolecules and their cognate receptors.

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