Toward Automated Synthesis of Oligosaccharides and Glycoproteins

Sears, P.S.; Wong, Chi‐Huey

doi:10.1126/science.1058899

Cited by 459 publications

(225 citation statements)

References 61 publications

Supporting

Mentioning

220

Contrasting

Unclassified

Order By: Relevance

“…Therefore, the development of enzymic approaches is of practical interest, and genetic modification has been extensively used to modify enzymic activity, to gain deeper understanding of catalytic mechanisms and to increase protein secretion. This is especially important as synthesis of specific carbohydrate derivatives at industrial scale by chemical methods is complex and requires protection and deprotection steps due to the presence of several hydroxyl groups of similar reactivity (Sears & Wong, 2001). …”

Section: Discussionmentioning

confidence: 99%

A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

Dagher

Bruno-Bárcena

2016

Microbiology

View full text Add to dashboard Cite

The b-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the a mating factor (MFa) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MFa (MFa-rBht (23-594) ), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53-and 14-fold, respectively. Leader interference was demonstrated when MFa preceded the putative classical rBHT leader (amino acids 1-22), explaining the limited secretion of soluble protein by P. pastoris (GS115 : : MFa-rBht (1-594) ). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-b-galactosidase single-chain variable antibody fragment scFv13R4.The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : MFa-rBht (23-594) -HIS and GS115 : : MFa-rBht ) showed comparable activity rates of GOS generation.

show abstract

Section: Discussionmentioning

confidence: 99%

A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

Dagher

Bruno-Bárcena

2016

Microbiology

View full text Add to dashboard Cite

show abstract

“…The accepted pathway for the biosynthesis of the sLe x and sLe a structures starts by sialylation of the type-2 and type-1 chain precursors, respectively, followed by fucosylation of the sialylated intermediates. [17][18][19][20][21] In embryogenesis, sialylation is an early phenomenon that appears at the beginning of cell differentiation and precedes fucosylation. 22 The step of fucosylation is then rate-limiting for the formation of sLe x/a in many different cell types and tissues.…”

Section: Discussionmentioning

confidence: 99%

Relationship of Sialyl-Lewisx/a Underexpression and E-Cadherin Overexpression in the Lymphovascular Embolus of Inflammatory Breast Carcinoma

Alpaugh

Tomlinson

Yin

et al. 2002

The American Journal of Pathology

View full text Add to dashboard Cite

Inflammatory breast carcinoma (IBC) isOur screening methods for certain types of breast cancer such as inflammatory breast cancer (IBC) have not impacted the disease's age-adjusted 5 year mortality because these types of breast cancer do not remain organconfined before they are detected. 1 One telltale sign of lack of organ confinement is the presence of lymphovascular invasion, a finding that is most pronounced in IBC. 2 Lymphovascular invasion is defined by tumor emboli within lymphovascular spaces. 3 These emboli have a unique microscopic appearance of compact clumps of tumor cells retracted away from the surrounding endothelial cell layer lining the lymphovascular space. The molecular determinants of this phenotype remain undefined. Recently our laboratory established a human SCID model of IBC (MARY-X) that exhibited florid lymphovascular tumor emboli in vivo and compact spheroids in vitro. 4,5 We have characterized, in part, the molecular basis of the IBC phenotype using this model. Our initial studies indicated that MARY-X overexpressed both Ecadherin and MUC1. 4 We chose to focus on E-cadherin initially and demonstrated that both the tumor cell emboli and the in vitro spheroids formed on the basis of an intact and overexpressed E-cadherin/␣,␤-catenin axis that mediated tumor cell adhesion analogous to the embryonic blastocyst. 5 Disruption of the E-cadherin/␣,␤-catenin axis with either anti-E-cadherin antibodies or E-cadherin dominant-negative mutant approaches caused complete disadherence of the tumor emboli in vivo. Furthermore in addition to these two manipulations, trypsin proteolysis and Ca ϩϩ depletion caused complete disadherence of the spheroids in vitro. In the present study we decided to investigate the role of overexpressed MUC1 and a possible mechanism that might explain the apparent lack of binding of the tumor embolus to the surrounding endothelium.

show abstract

“…1A) (9). However, a detailed analysis of the structural features that hasten folding and increase protein stability has not yet been conducted because of the challenge of assessing folding in vivo (9) and the difficulty of expressing and/or synthesizing homogeneous glycoproteins for in vitro studies (10,11).…”

mentioning

confidence: 99%

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

Hanson

Culyba

Hsu

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

215

203

View full text Add to dashboard Cite

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the ␤-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycan's first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc 2, improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.glycoprotein stability ͉ N-glycan function ͉ N-glycosylation ͉ protein folding ͉ kinetics

show abstract

Toward Automated Synthesis of Oligosaccharides and Glycoproteins

Cited by 459 publications

References 61 publications

A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

A novel N-terminal region of the membrane β-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

Relationship of Sialyl-Lewisx/a Underexpression and E-Cadherin Overexpression in the Lymphovascular Embolus of Inflammatory Breast Carcinoma

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

Contact Info

Product

Resources

About