ScFv antibody-induced translocation of cell-surface heparan sulfate proteoglycan to endocytic vesicles: evidence for heparan sulfate epitope specificity and role of both syndecan and glypican.
Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.
Section of Oncology, Department of Clinical Sciences, Lund University, SE-221 85 Lund, Sweden. anders.wittrup@med.lu.se
Anders
A
Wittrup
Si-He
SH
Zhang
Gerdy B
GB
ten Dam
Toin H
TH
van Kuppevelt
Per
P
Bengtson
Maria
M
Johansson
Johanna
J
Welch
Matthias
M
Mörgelin
Mattias
M
Belting
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D000939:Epitopes / Q000737:chemistry*; D053673:Glypicans / Q000737:chemistry*; D015488:HIV Antigens / Q000737:chemistry; D006367:HeLa Cells; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry; D019151:Peptide Library; D053667:Syndecans / Q000737:chemistry*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry
D000939:Epitopes; D053673:Glypicans; D015488:HIV Antigens; D019812:Heparan Sulfate Proteoglycans; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D053667:Syndecans; D054322:tat Gene Products, Human Immunodeficiency Virus