Highly oriented recombinant glycosyltransferases: site-specific immobilization of unstable membrane proteins by using Staphylococcus aureus sortase A.
Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides, and various artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both in the basic studies of their functional roles in various biological processes and in the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that the immobilization of extremely unstable membrane-bound glycosyltransferases on some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate a versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the first time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta1,4-galactosyltransferase or recombinant Helicobacter pylori alpha1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically immobilized enzymes exhibited the desired sugar transfer activity, an improved stability, and a practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, as well as glycosyltransferases are unstable and highly oriented membrane proteins, the merit of our strategy based on "site-specific" transpeptidation is evident because the reaction proceeds only at an engineered C-terminus without any conformational influence around the active sites of both enzymes as well as heptad repeats of rHFucT required to maintain native secondary and quaternary structures during the dimerization on cell surfaces.
Ito
Takaomi
T
Graduate School of Life Science and Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Sadamoto
Reiko
R
Naruchi
Kentaro
K
Togame
Hiroko
H
Takemoto
Hiroshi
H
Kondo
Hirosato
H
Nishimura
Shin-Ichiro
S
D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005647:Fucosyltransferases / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology; D006801:Humans; D016256:Lewis X Antigen / Q000096:biosynthesis / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
D000588:Amines; D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D016256:Lewis X Antigen; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012685:Sepharose; D019881:Aminoacyltransferases; C120542:sortase A; D016695:Glycosyltransferases; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D009237:N-Acetyllactosamine Synthase; D003546:Cysteine Endopeptidases