CELL-SPECIFIC TARGETING OF FUSION PROTEINS THROUGH HEPARIN BINDING - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 402

are highly electrostatic through positively charged amino
acid sequences that bind to the strongly anionic HS; and
occurs with a diverse repertoire of proteins including growth
factors, cytokines, chemokines, cell-surface receptors,
matrix components, enzymes, and viruses (for review see
[29]). As many of these HS-binding proteins are character-
ized and purified by affinity chromatography on columns of
heparin, they are often collectively referred to as heparin-
binding proteins, even though the endogenous binding part-
ners are HSPGs.
While not used for targeting, one of the best-studied
examples of a heparin-binding protein is antithrombin [30].
Heparin is used therapeutically to bind to antithrombin and
results in the inactivation of thrombin, Factor Xa, and other
members of the coagulation cascade. High-affinity binding
requires a specific pentasaccharide within heparin that inter-
acts with positively charged amino acids on the protein. The
subsequent conformational change accelerates its binding
and inhibition of the proteases involved in coagulation.
Perhaps, the most extensively studied heparin-binding
growth factor is fibroblast growth factor (FGF). FGFs are a
large family of monomeric growth factors that function in
cell growth, angiogenesis, wound healing, and embryonic
development. Heparin-binding forms of FGF bind HS not
only for tissue targeting but also to enhance their signaling
through high-affinity membrane receptors [31]. A tissue-
binding assay has been developed that shows that specific
forms of FGF bind selectively to some tissue and cells and
not others based on their HS composition [32]. In addition to
localizing FGF in various tissue locations, interactions with
HSPGs significantly enhance signal transduction by low-
ering the concentration of FGF required for signaling initia-
tion and extending the response duration. There is evidence
to suggest that HS further activates signaling by forming a
bridging complex between FGF and its receptor, that also
has a heparin-binding site, to produce a ternary complex that
promotes FGF signaling [33].
In addition to targeting and enhancing receptor activa-
tion, proteins bound to HS are protected from proteolytic
degradation. HS has been shown to protect cytokines from
endogenous proteolytic cleavage during the inflammatory
process so that they can continue to carry out their important
roles [34]. Interferon- g (IFN- g ) is a cytokine that is critical
for innate and adaptive immunity against viral and intra-
cellular bacterial infections. It binds with high affinity to HS
and heparin molecules through its carboxyl-terminal
domain. Unbound IFN-gamma is cleaved rapidly at the
carboxyl-terminal side, resulting in removal of at least 18
amino acids and inactivation of the cytokine. When bound to
heparin, the plasma clearance of IFN-gamma is decreased
greatly and the extent of its carboxyl-terminal domain
degradation is limited to less than 10 amino acids, giving
rise to sixfold increases of the cytokine activity. This
resistance to proteolysis and resultant longer half-life of

heparin-binding proteins is an attractive feature that could be
incorporated into engineered biopharmaceuticals that bind
heparin.
In addition to using heparin-binding proteins as receptor
agonists, nature has also used heparin-binding capability to
produce targeted antagonists. An example comes from the
Wnt signaling pathway [35]. Wnt proteins are a large family
of structurally related secreted heparin-binding glycopro-
teins that mediate fundamental biological processes such as
cell polarity and proliferation, tissue patterning, and tumor-
igenesis. The Wnt signaling is initiated by the binding of
Wnt proteins to a membrane receptor complex consisting of
the Frizzled (Fz) family and members of the low-density
lipoprotein receptor-related family. HS augments the action
of secreted Wnt antagonists, some of which are also heparin-
binding proteins. An example is the secreted frizzled-related
protein-1 (sFRP-1) that belongs to a class of extracellular
antagonists that modulate Wnt signaling pathways by pre-
venting ligand-receptor interactions between the Wnt and
the Frizzled membrane receptor. Heparin can specifically
enhance recombinant sFRP-1 accumulation in a cell type-
specific manner that requires O-sulfation in heparin and FGF
signaling. Moreover, it seems that heparin also directly
stabilizes the sFRP-1 protein in vitro.

27.5 VIRUSES TARGET CELLS THROUGH
HEPARIN BINDING

It is not surprising that if nature has created a system for
tissue-specific targeting of proteins using HSPGs that
viruses have evolved to take full advantage of this system.
In fact, a number of viruses use HSPGs as their specific
targets for viral entry that lead to tissue-specific infectivity
[36]. Herpes simplex virus type 1 (HSV-1) binds to cells
through interactions of viral glycoprotein D with cell-surface
HS chains consisting 3-O-sulfation (3-OS) generated by 3-O
sulfotransferase (3OST). These interactions promote some
selectivity in targeting HSV-1 infections to specific cell
populations such as the dorsal root ganglion cells where
the virus can lay dormant for long periods. There are six
isoforms of 3OST in humans [37,38]. 3OST-2, -3, and -4
have been reported to make cells susceptible to HSV-1 entry
and produce two disaccharides, one of which is the tetra-
sulfated disaccharide (Di-tetraS) unit [39]. While the 3OST-
3 transcripts are highly expressed in spleen and liver, the
3OST-2 and -4 are expressed specifically in brain. Consis-
tently, Di-tetraS is relatively highly expressed in spleen and
liver. Although the interaction does not suffice for viral
entry, bound viruses can then interact with other host
determinants for internalization.
Another family of viruses that use HSPGs for cell
recognition are the adeno-associated virus type 2 (AAV-
2). Mutagenesis experiments of AAV-2 capsid proteins