Synthesis and use of novel fluorescent glycosphingolipids for estimating β-glucosidase activity in vitro in the absence of detergents and subtyping Gaucher disease variants following administration into intact cells

Vered Agmon, Sara Cherbu, Arie Dagan, Marie Grace, Gregory A. Grabowski, Shimon Gatt

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and LRO-trihexosylceramide (LRO-THC) were synthesized and utilized for estimating activities of the lysosomal, acid β-glucosidase in cell extracts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolipids is the fact that a fluorescent probe (lissamine rhodamine) is linked in a sulfonylamide linkage to the sphingosyl residue of the sphingolipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid β-glucosidase in vitro was the observation that enzymatic hydrolysis occurs in the absence of detergents and that hydrolytic rates are, in fact, reduced in the presence of Triton X-100 and/or sodium taurocholate. Also, both glycolipids penetrated the membrane of intact fibroblasts in the absence of serum and were hydrolyzed in lysosomes of the intact cells. The rates of intracellular hydrolysis decreased with the severity of the Gaucher disease subtypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceramide to LRO-glucosylceramide was an indicator for the specific GD-subtype.

Original languageEnglish
Pages (from-to)72-79
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1170
Issue number1
DOIs
StatePublished - 29 Sep 1993
Externally publishedYes

Keywords

  • (Human)
  • Fibroblast
  • Gaucher disease
  • Glycosphingolipid
  • β-Glucosidase activity

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