CELL-SPECIFIC TARGETING OF FUSION PROTEINS THROUGH HEPARIN BINDING - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 400

27.2 HEPARAN SULFATE STRUCTURE AND
BIOSYNTHESIS CREATE DIVERSITY AND A
TEMPLATE FOR TARGETING SPECIFICITY

tetrasaccharide. This is catalyzed by enzymes that belong
to the exostosis family of proteins (EXT) [13]. As the chain
polymerizes, it undergoes a series of modifying enzymatic
reactions that generate further heterogeneity of HS struc-
ture as well as give HSPGs their strongly negative electro-
static charge. These modifications include GlcNAc
N-deacetylation/N-sulfation catalyzed by N-deacetylase/
N-sulfotransferase (NDST) family of enzymes, epimeriza-
tion of glucosamine to iduronic acid, 2-O-sulfation, and
sulfation of GlcNAc by 6-O-sulfotransferases (6OST) or 3-
O-sulfotranserases (3OST). The six potential modifica-
tions on every disaccharide lead to a great level of
sequence diversity. Twenty-three unique disaccharides
out of as many as 48 different disaccharides that can occur
theoretically have thus far been identified from vertebrates
[12]. The net result of this biosynthetic process is wide
diversity of HS chains with domains of contiguous dis-
accharides containing N-sulfation, O-sulfation, and idur-
onic acid interspersed by variable tracts of unmodified
domains. Not surprisingly, the binding sites for HS-inter-
acting ligands, such as growth factors, occur in highly
modified segments or at the borders between modified and
unmodified HS segments [14].
The complexity and diversity of sugar structures does
not end with biosynthesis. Further processing occurs once
the HSPG reaches the plasma membrane, often as a means
to carry out a biological process. Specific sulfate groups
can be removed from the HS chains by plasma membrane-
bound endosulfatases. For example, Sulf1 and Sulf2 are
extracellular HSPG-specific sulfatases that catalyze the
removal of sulfate from the C-6 position of glucosamine
[15,16]. The GPI anchors of glypicans are subject to
cleavage to shed the HSPGs from the cell surface [17].
Core proteins can also undergo proteolytic cleavage to be
released into the ECM. For example, Syndecan-1 and -4
are expressed on epithelial cells in the lung [18]. Upon
inflammation, they are released into the bronchoalveolar
fluid mediated by the disintegrin-like metalloproteinase
ADAM17. Extracellular heparanase is an endo- b - D -glucu-
ronidase that cleaves HS side chains at certain sites [19].
They are strongly implicated in structural remodeling of
the ECM as a consequence of HS cleavage. They can
regulate growth factor and/or chemokines signaling by
selectively mobilizing HS-bound ligands [20].
In summary, the various core proteins and essentially
limitless combinations of distinct sugar compositions and
modifications that occur during biosynthesis and postassem-
bly processing contribute to an enormous structural diversity
of HSPGs. The key to this structural heterogeneity is to use it
in specific biological contexts and in segregated ways that
are closely linked to the proteins that interact with the
various HS structures. Examples of how these are used in
nature and how one can harness this diversity for drug
targeting are discussed later in this chapter.

To be a reliable biopharmaceutical target, HSPGs need to
provide sufficient diversity and specificity for distinct bio-
logical contexts. Heparin, the well-known anticoagulant
drug, is actually a processed and highly sulfated form of
HS. While heparin is found mostly in connective-tissue type
mast cells, HSPGs are found ubiquitously and abundantly on
virtually every cell surface and in the ECM of most tissues.
The diversity of HSPGs results first from a large number of
core proteins that serve to distribute them to different regions
of the extracellular space and second from the enormous
variety of negatively charged, sulfated HS structures (See
Figure 27.1 for structural schematics).
Most cells bear multiple types of proteoglycan core
proteins. HSPGs can be generally categorized into three
major classes based on the core protein primary structure
[8-10]. Matrix HSPGs are secreted into ECM and include
perlecan, agrin, and collagen XVIII. Syndecans are type I
transmembrane HSPGs that carry the HS chains on their
extracellular domains near the N-terminus. Most verte-
brates have four syndecan genes, whereas all invertebrates
have only one. Glypicans are the third class of HSPGs that
are also anchored on the cell surface, but through a
glycophosphatidylinositol linkage. Vertebrates typically
have six glypican genes, whereas invertebrates only
have two. The extracellular domains of betaglycan and a
splice variant of CD44 (v3) are also subject to HS attach-
ment. Each core protein often has several attachment sites
capable of bearing multiple GAG chains. For example, the
glypicans typically contain up to three HS side chains.
Additionally, there are a numberofproteoglycansthathave
more than one type of GAG chain, creating diversity of
structure even within the same protein backbone. For
example, neuropilin-1 is a proteoglycan that has combina-
tions of HS, CS, and DS [11].
Besides the large number of core proteins available for
HS decoration, HS itself is one of the most complex
biomolecules. After a core protein is translated, many
membrane-bound glycotransferases and modifying
enzymes in the endoplasmic reticulum and the Golgi
apparatus function in a specific and sequential manner
to assemble HS chains [12]. HS synthesis initiates with
the transfer of xylose to specific serine residues in a
consensus sequence of serine-glycine with one or more
flanking acidic residues. Attachment of two D -galctose
(Gal) residues and a glucuronic acid (GlcA) completes
the formation of the core-protein linkage tetrasaccharide.
The formation of the linkage tetrasaccharide is identical in
CS, DS, and HS. HS chains elongate by consecutive
addition of alternating glucuronic acid (GlcA) and N-
acetylglucosamine

(GlcNAc) onto the linkage