Biochemical characterization of fluorescent-labeled recombinant human alpha- l -iduronidase in vitro

Brigette L. Tippin, Larisa Troitskaya, Shih Hsin Kan, Amanda K. Todd, Steven Q. Le, Patricia I. Dickson

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-l-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 M. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

Original languageEnglish
Pages (from-to)391-396
Number of pages6
JournalBiotechnology and Applied Biochemistry
Volume58
Issue number6
DOIs
StatePublished - Nov 1 2011
Externally publishedYes

Keywords

  • Hurler
  • enzyme replacement therapy
  • fluorescence
  • iduronidase
  • lysosomal
  • mucopolysaccharidosis

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