title: Further studies on the site-specific protein modification by microbial transglutaminase.
abstract: A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.
authors.affiliation: Ajinomoto Company Inc., Pharmaceutical Research Laboratories, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, 210-8681, Japan. haruya_satou@ajinomoto.com
authors.forename: H
authors.initials: H
authors.lastname: Sato
authors.forename: E
authors.initials: E
authors.lastname: Hayashi
authors.forename: N
authors.initials: N
authors.lastname: Yamada
authors.forename: M
authors.initials: M
authors.lastname: Yatagai
authors.forename: Y
authors.initials: Y
authors.lastname: Takahara
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D004337:Drug Carriers / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics; D005690:Galactose / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D006031:Glycosylation; D007376:Interleukin-2 / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism* / Q000493:pharmacokinetics; D008099:Liver; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity; D010449:Peptide Mapping; D011092:Polyethylene Glycols; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004337:Drug Carriers; D007376:Interleukin-2; C067245:interleukin-2, polyethylene glycol-modified; D011092:Polyethylene Glycols; D011503:Transglutaminases; D005690:Galactose