Question: Based on the article below,write a review of biological functions asso

Question

Question: Based on the article below,write a review of biological functions associated with proteoglycans.

(THE FIGURES WERE NOT PROVIDED!!)

Title: Proteoglycans Form and Function Introduction

It has been nearly 20 years since the original

publication of a comprehensive classification of

proteoglycan gene families [1]. For the most part,

these classes have been widely accepted. However,

a broad and current taxonomy of the various

proteoglycan gene families and their products is not

available. In contrast to the classification of glycosaminoglycans

(GAGs), primarily based on the chemical

structure of their repeating disaccharide units,

classifying proteoglycans is a much more complex

task [2]. We propose a comprehensive and simplified

nomenclature of proteoglycans based on three criteria

including: Cellular and subcellular location, overall

gene/protein homology, and the presence of specific

protein modules within their respective protein cores.

Whereas the first two attributes have been utilized in

the past for various nomenclatures, the third attribute

is ofmore recent development and represents a sort of

“intrinsic signature” for various protein cores. Indeed,

modular design is based on the simple concept that

protein cores are made up of finite units, like pieces

of Lego. The units represent a minimum level of

organization and a module can be thought of as a

functional domain that affects cell–matrix dynamics.

Another key feature is that each module/functional

unit can be stable and can fold on its own, without

being part of the large precursor protein. Thus, a

module is a self-contained component. An example of

this is the LG3 domain of endorepellin, the C-terminal

globular-like domain of perlecan, which has recently

been crystallized [3]. Below, we will critically assess

the field of proteoglycans which now encompass forty

three distinct genes and a much higher number of

proteoglycans due to alternative splicing, thereby

providing a very rich and biologically-active group of

molecules. As hyaluronan and the enzymes involved

in the synthesis and degradation of various GAGs are

not covered in this review, readers are referred to

recent reviews covering these closely-related subjects

General features

Four major proteoglycan classes encompass

nearly all the known proteoglycans of the mammalian

genome (Fig. 1). Observing the types of

proteoglycans based on cellular and subcellular

localization, we can see that there is only one

intracellular proteoglycan, serglycin. This unique

proteoglycan forms a class on its own as it is the only

proteoglycan that carries heparin side chains.

Serglycin is packaged in the granules of mast cells

and serves as biological glue for most of the

intracellular proteases stored within the granules

[19] . Another general observation is that heparan

sulfate proteoglycans (HSPGs) are prevalently

associated with the cell surface or the pericellular

matrix. The HSPGs are intimately associated with

the plasmamembranes of cells, either directly via an

intercalated protein core or via a glycosyl-phosphatidyl-

inositol (GPI) anchor, and function as

major biological modifiers of growth factors such

as FGF, VEGF and PDGF among others. Similar

functions are also performed by the HSPGs located

in the basement membrane zone, in addition to their

ability to interact with each other and with key

constituents of the basement membrane, including

various laminins, collagen type IV, and nidogen.

Presentation of growth factors to their cognate

receptors in a biologically-favorable form is a major

function of cell surface and pericellular HSPGs.

Another key role is participating in the generation

and long range maintenance of gradients for

morphogens during embryogenesis and regenerative

processes.

As we move away from the cells in a centrifugal

manner, chondroitin- and dermatan sulfate-containing

proteoglycans (CSPGs and DSPGs, respectively)

predominate. These proteoglycans function as

structural constituents of complex matrices such as

cartilage, brain, intervertebral discs, tendons and

corneas. Thus, among other functions, they provide

viscoelastic properties, retain water and keep

osmotic pressure, dictate proper collagen organization

and are the main molecules responsible for

corneal transparency. The extracellular matrix also

contains the largest class of proteoglycans, the

so-called small leucine-rich proteoglycans (SLRPs)

which are themost abundant products in terms of gene

number. These SLRPs can function both as structural

constituent and as signaling molecules, especially

when tissues are remodeled during cancer, diabetes,

inflammation and atherosclerosis. SLRPs interact with

several receptor tyrosine kinases (RTKs) and Toll-like

receptors, thereby regulating fundamental processes

including migration, proliferation, innate immunity,

apoptosis, autophagy and angiogenesis. Below we

will discuss the rationale for grouping certain proteoglycans

in the same class and their overall biological

function.

Intracellular proteoglycans

It is quite amazing that since the original cloning of

serglycin, the first proteoglycan-encoding gene to be

sequenced, no other true intracellular proteoglycan

has been discovered. Serglycin occupies a class of

its own insofar as it is the only proteoglycan that is

covalently substituted with heparin due to its

consecutive (and quite unique) Ser-Gly repeats,

essentially a silk-like sequence. Serglycin has been

utilized primarily by mast cells for the proper

assembly and packaging of the numerous proteases

that are released upon inflammation [19] . The

defects in the formation of mast cell granules

observed in Srgn/ mice are remarkably similar to

those observed in mast cells derived from mice

lacking N-deacetylase/N-sulfotransferase 2, a key

enzyme involved in the sulfation of heparin [19] .

Thus, serglycin promotes granular storage via

electrostatic interaction between its highly-anionic

heparin chains and basic residues within the various

proteases of the secretory granules. It is becoming

evident, however, that all inflammatory cells express

serglycin and store it within intracytoplasmic granules

where, in addition to proteases, serglycin binds

and modulates the bioactivity of several inflammatory

mediators, chemokines, cytokines and growth

factors [20] .

More recently, serglycin has been found in a wide

variety of non-immune cells such as endothelial

cells, chondrocytes and smooth muscle cells [21] .

Cell-surface serglycin promotes adhesion of myeloma

cells to collagen I and affects the expression ofMMPs

[22] . These findings have been corroborated by in vivo

studies where serglycin knockdown attenuates the

multiple myeloma growth in immunocompromised

mice [23] . It has been proposed that some of these

effects are mediated by a specific interaction between

serglycin and cell-surface CD44 [23] , a known

receptor for hyaluronan [24,25] . It has been recently

shown that serglycin is a key component of the cell

inflammatory response in activated primary human

endothelial cells as both LPS and IL-1 increase its

synthesis and secretion [26] . Notably, serglycin can

be substituted with chondroitin sulfate (CS), and in

several circulating cells serglycin contains lower

sulfated CS-4 chains [21] . In contrast, several

hematopoietic cells (mucosal mast cells, macrophages

etc.) express serglycin with highly sulfated

CS-E. Although the significance of this phenomenon

is not fully appreciated, it is likely that these isoforms

of serglycin might have different functions in a

cell-context specific manner. Serglycin is a marker of

immature myeloid cells and interacts with many

bioactive components including histamine, TNF-

and proteases [27] . In general, serglycin expression

correlates with a more aggressive malignant phenotype

and it has been recently proposed that serglycin

protects breast cancer cells from complement attack,

thereby supporting cancer cell survival and progression

[28] .

Cell surface proteoglycans

In this class, there are thirteen genes, seven

encoding transmembrane proteoglycans and six

encoding GPI-anchored proteoglycans. With the

exception of two gene products, NG2 and phosphacan,

all contain heparan sulfate side chains.

Syndecans

The eponym syndecan was coined by the late

Merton Bernfield [29] to define a class of transmembrane

proteoglycans that would connect (from the

Greek syndein, “bind together”) the surface of the cells

to the underlying extracellular matrix. The syndecan

family now comprises four distinct genes encoding

single-pass transmembrane protein cores which

include an ectodomain, a transmembrane region

and an intracellular domain [4,30] (Fig. 2). The

ectodomains exhibit the lowest amount of amino

acid sequence conservation, no more than 10–20%,

in contrast to the transmembrane and cytoplasmic

domains which are 60–70% identical. A recent study

has shown that the ectodomain of syndecans is

natively disordered and this characteristic allows

syndecans to interact with a variety of proteins and

ligands, thereby providing enrichment in their biological

function [31]. The ectodomain contains the GAG

attachment sites, which are often covalently-linked to

HS and sometimes to CS, making syndecans hybrid

proteoglycans. Several cell types shed syndecan into

the pericellular environment through the action of

MMPs. For example, it has recently been shown that

shed syndecan-2 retards angiogenesis by inhibiting

endothelial cell migration [32], a key step in neovascularization

[33]. The transmembrane domain contains

a dimerization motif (GxxxG) that mediates both

homo-dimerization and hetero-dimerization [30]. The

intracellular domain is composed of two regions of

conserved amino acid sequence (C1 and C2),

separated by a central variable sequence of amino

acids that is distinct for each family member (V) [34].

Notably, the C-terminus of all the four syndecans

harbors a unique signature (EFYA) that binds

PDZ-containing proteins. Generally, PDZ-containing

proteins contribute to a proper anchor of transmembrane

proteins to the cytoskeleton, thereby holding

together large signaling complexes.

Syndecans are involved in a wide variety of

biological functions, too vast to be reviewed here,

but reviewed recently [5,30,34]. Briefly, syndecans

bind numerous growth factors, especially through

their HS chains, and dictate morphogen gradients

during development. In concert with other cellsurface

HSPGs, syndecans can act as endocytosis

receptors and are also involved in the uptake of

exosomes [35]. Syndecans play key roles as

co-receptors for many RTKs and can also function

as receptors for atherogenic lipoproteins [36].

Indeed, there is strong genetic evidence that

syndecan-1 is the main HSPG mediating clearance

of triglyceride-rich lipoproteins derived from either

the liver or from intestinal absorption [37].

Many, if not all the syndecans, can also act as

soluble HSPGs via partial proteolysis of their

juxtamembrane region releasing their whole ectodomains.

This shedding is considered a powerful

post-translational modification that can regulate the

amount of HSPG linked to the cell surface and

that present in the pericellular microenvironment

[30]. Several inflammatory cytokines can induce

syndecan shedding by triggering outside-in signaling

and by activating several metalloproteinases. In the

case of hepatocytes, shedding of syndecan-1 occurs

via PKC-dependent activation of ADAM17, and

this impairs VLDL catabolism and promotes hypertriglyceridemia

[38] . Importantly, soluble syndecan-1

promotes the growth of myeloma tumors in vivo [39] ,

and this process, i.e. the shedding of syndecan-1, is

enhanced by heparanase [40] , thereby offering a

novel mechanism for promoting cancer growth and

metastasis [41,42] . Notably, chemotherapy stimulates

syndecan-1 shedding, a potential drawback of the

treatment that could potentially favor tumor progression

[43] . The biological interplay between heparanase-

evoked shedding of syndecan-1 and myeloma

cells leads to enhanced angiogenesis [44] , further

supporting cancer growth. As mentioned above,

however, shed syndecan-2 inhibits angiogenesis via

a paracrine interaction with the protein tyrosine

phosphatase receptor CD148, which in turn deactivates

1-containing integrins [32] , presumably 1 1

and 2 1, two main angiogenesis receptors. In

contrast, the ortholog syndecan-2 is required for

angiogenic sprouting during zebrafish development

[45] .

An emerging new role for syndecan-1 is linked to

its ability to reach the nuclei in a variety of cells. Initial

observations showed that myeloma and mesothelioma

cells contain syndecan-1 in their nuclei [46,47]

and this nuclear translocation is also regulated by

heparanase [46] , indicating that there must be a

cellular receptor for shed syndecan-1 that could

mediate its nuclear targeting and transport. In

support of these studies are previous observations

that exogenous HS can translocate to the nuclei and

modulate the activity of DNA Topoisomerase I [48]

and histone acetyl transferase (HAT) [49] . N-terminal

acetylation of histones by HAT is linked to transcriptional

activation, and this process is finely tuned by its

counteracting enzyme, histone deacetylase (HDAC).

Heparanase-evoked loss of nuclear syndecan-1

causes an increase in HAT enzymatic activity and

enhances transcription of pro-tumorigenic genes [50] .

Syndecan-1 that is shed from myeloma tumor cells is

uptaken by bone marrow stromal cells and is

transported to the nuclei by amechanismthat requires

its HS chains, as this process is inhibited by heparin

and chlorate [51] . Once nuclear, soluble syndecan-1

binds to HAT p300 and inhibits its activity, thereby

providing a new mechanism for tumor– host cell

interaction and cross-talk [52] .

CSPG4/NG2

The melanoma-associated chondroitin sulfate proteoglycan

(MCSP) was discovered over 30 years

ago as a transmembrane proteoglycan and a highly

immunogenic tumor antigen ofmelanoma tumor cells.

This proteoglycan has been subsequently detected in

various species, with many names designating the

same gene product. The rat ortholog of MCSP is

called nerve/glial antigen 2 (NG2) [53] , while the term

CSPG4 designates the human gene. We will use

CSPG4/NG2 terminology with the idea that some of

the functional properties have not been fully described

in the human and rat species [54] . CSPG4/NG2 is a

single-pass, type I transmembrane proteoglycan

carrying one chondroitin sulfate chain, and harboring

a large ectodomain composed of three subdomains

(Fig. 2 ). The N-terminal domain (D1 subdomain)

contains two laminin-like globular (LG) repeats. It is

likely that the LG domains as in other proteoglycans

(i.e. perlecan and agrin, see below) mediate ligand

binding, cell– matrix and cell– cell interactions, as well

as interaction with integrins and receptor tyrosine

kinase (RTK). The central subdomain D2 contains 15

tandem repeats of a new module called CSPG [54] .

The CSPG repeat is a cadherin-like and tumorrelevant

module which is predicted to be involved in

cell– matrix interaction, further modulated by the CS

chain covalently attached to this module. Indeed,

CSPG modules bind to collagens V and VI, FGF and

PDGF. The juxtamembrane subdomain D3 contains a

carbohydrate modification able to bind integrins and

galectin, as well as numerous protease cleavage

sites. Accordingly, the intact ectodomain and fragments

thereof can be detected in sera from normal

and melanoma-carrying patients [54] . The transmembrane

domain of CSPG4/NG2 is quite interesting

insofar as it has a unique Cys residue, generally not

found in transmembrane regions. The intracellular

domain harbors a proximal region with numerous Thr

phospho-acceptor sites for PKC and ERK1/2, and a

distal region encompassing a PDZ-binding module

similar to the syndecan family. The latter can bind to

the PDZ domain of several scaffold proteins involved

in intracellular signaling, including syntenin, MUPP1

and GRIP1.

Functionally, CSPG4/NG2 proteoglycan promotes

tumor vascularization [55] and because of its

predominant perivascular localization, CSPG4/NG2

may modulate the availability of FGF at the cell

surface as well as the bioactivity and signal

transduction of FGF receptors [56] . This CSPG

binds to collagen VI in the tumor microenvironment

and promotes cell survival and adhesion via the

PI3K pathway [57] . Indeed, targeting CSPG4/NG2 in

two animal models of highly-malignant brain tumors

reduces tumor growth and angiogenesis [58] .

Moreover, a combinatorial treatment using activated

natural killer cells and a monoclonal antibody toward

CSPG4/NG2 is capable of eradicating glioblastoma

xenografts more efficiently than single therapies

[59] .

It has recently been discovered that NG2 controls

the directional migration of oligodendrocyte precursor

cells by constitutively stimulating RhoA GTPases

[60]. Based on NG2 ability to regulate adhesion

RhoA GTPase and growth factor activities, it is likely

that this transmembrane proteoglycan might play a

key role in regulating cell polarity in response to

extracellular cues [61] .

Perdido/Kon-tiki , the Drosophila ortholog of mammalian

CSPG4 , genetically interacts with integrins

during Drosophila embryogenesis, and its loss is

embryonic lethal [62] . RNAi-mediated suppression

of Perdido/Kon-tiki in the muscles, just before adult

myogenesis starts, induces misorientation and

detachment of Drosophila adult abdominal muscle,

generating a phenotype similar to the embryonic

lethal ones [63] . Thus, it is possible that, based on its

high conservation through species, mammalian

CSPG4 could also play a role in myogenesis and

function as well.

A recent study has added another function to

CSPG4 by involving this cell surface proteoglycan in

the pathogenesis of severe pseudomembranous

colitis. CSPG4 acts as a receptor for the Clostridium

difficile toxin B, one of the key toxins secreted by this

gram-positive and spore-forming anaerobic bacillus

[64] . The interaction occurs between the N-terminus

of CSPG4 and the C-terminus of toxin B. This

discovery, if confirmed in future studies, opens new

therapeutic targets for the treatment of this severe

and often lethal form of enterocolitis.

Betaglycan/TGF type III receptor

In 1991, two back-to-back papers reported on the

isolation and cloning of a membrane-anchored

proteoglycan with high affinity for TGF , and thus

named betaglycan [65,66] . Betaglycan, also known as

TGF type III receptor (TGFB3), is a single-pass

transmembrane proteoglycan that belongs to the

TGF superfamily of co-receptors (Fig. 2 ). The

extracellular domain contains several potential GAG

attachment sites and protease-sensitive sequences

near the plasma membrane. The short intracellular

domain is highly enriched in Ser/Thr (N 40%) and

some of these residues are candidate sites for

PKC-mediated phosphorylation [65] . Betaglycan

amino acid sequence is highly similar to that of

endoglin, a close member of the same superfamily.

The membrane-proximal ectodomain of betaglycan

contains a unique module called zona pellucida

(ZP)-C [67] . The ZP module is a structural element

typically found in the ectodomain of eukaryotic

proteins composed of a Cys-rich bipartite structure

joined by a linker. Generally, proteins harboring ZP

modules tend to polymerize and assemble into long

fibrils of specialized extracellular matrices [67] . In the

case of betaglycan and endoglin these ZP modules

are not utilized for polymerization, rather they function

as membrane co-receptors for the TGF superfamily

members [68] . The intracellular domain contains a

PDZ-binding element similar to that observed in the

syndecan family of proteoglycans (Fig. 1 ).

Betaglycan is a ubiquitously-expressed cell surface

proteoglycan that acts as a co-receptor for

members of the TGF superfamily of Cys knot

growth factors which also include activins, inhibins,

GDFs and BMPs [69,70] . For example, betaglycan

enhances the binding of all the TGF isoforms to the

signaling TGF complex [71] and is needed for

TGF 2 high-affinity interaction with the receptor

complex. Betaglycan also blocks the aggressiveness

of ovarian granulosa cell tumors by suppressing

NF- B-evoked MMP2 expression [72] .

Betaglycan, together with other TGF -binding

SLRPs, i.e. decorin and biglycan (see below), can

be cleaved by granzyme B, thereby releasing an

active form of TGF [73] . Ectodomain shedding of

betaglycan is indeed necessary for betaglycanmediated

suppression of TGF signaling and breast

cancer migration and invasion [74] . The ability of

betaglycan to affect epithelial mesenchymal transformation

[70] , together with genetic evidence of

embryonic lethality in Tgfbr3/ mice, suggests that

betaglycan may play a unique and non-redundant

function during development.

Another important feature of betaglycan is its

ability to modulate the subcellular topology of the

signaling receptor complex via its PDZ-binding

domain, which interacts with PDZ-containing proteins

such as -arrestin [75] . This interaction, as

well as that between betaglycan intracellular domain

and GIPC, would stabilize betaglycan at the

cell surface and potentiate its bioactivity. Finally,

betaglycan is involved in regulating many functions

including reproduction and fetal growth [75] , and is

a putative tumor suppressor in many forms of

cancer [76] . Several additional betaglycan-evoked

activities have been recently reviewed elsewhere

[75] .

Phosphacan/receptor-type protein tyrosine

phosphatase

Phosphacan, originally isolated from rat brain, is a

CSPG that interacts with neurons and neural

cell-adhesion molecules (N-CAM) and corresponds

to the soluble ectodomain of a Receptor-type protein

tyrosine phosphatase (RPTP ) [77] . The phosphacan

gene (PTPRZ1 ) encodes a single-pass type

I membrane protein with a relatively large ectodomain

harboring an N-terminal module homologous to

the alpha-carbonic anhydrase (Fig. 2 ). Distal to this,

there is a fibronectin type III domain. The ectodomain

contains six Ser-Gly repeats, at least four of

which are flanked by acidic residues suggesting

potential glycanation sites. Sporadically, phosphacan

can also be substituted with keratan sulfate

chains. Notably, alternative splice variants encoding

different protein isoforms have been described

but their full-length nature has not yet been

established.

Functionally, the ectodomain of phosphacan mediates

cell– cell adhesion by hemophilic binding. In

addition, phosphacan's ability to bind N-CAM and

tenascin in a calcium-dependent manner suggests

that RPTPs may also modulate cellular interactions

via heterophilic mechanisms [77] . Indeed, phosphacan

blocks the growth-promoting ability of N-CAM,

axonin-1 TAG-1 and tenascin, and is crucial in the

oriented movement of post-mitotic cells during cortical

development of the brain [78] .Moreover, phosphacan

binds contactin, another member of the Ig superfamily

like N-CAM, and the extracellular portion of the

voltage-gated sodium channel [79] . The latter interaction

appears to be mediated by the carbonic

anhydrase-like module of phosphacan's ectodomain.

It has been proposed that phosphacan, as an integral

extracellular matrix constituent of the neural stem cell

compartment, would contribute to the privileged

microenvironment that supports self-renewal and

maintenance of the neural stem cell niche [80] .

Glypicans/GPI-anchored proteoglycans

Glypicans (GPC) are HSPGs that are bound to

the plasma membrane via a C-terminal lipid moiety

known as GPI, for glycosylphosphatidylinositol,

linkage or anchor (Fig. 2 ). There are six independent

genes in the mammalian genome which can be

subdivided into two broad classes: GPC1/2/3/6 and

GPC3/5 with orthologs present across Metazoan

including Dally and Dlp in Drosophila melanogaster

[81] . Although most of the protein core is unique to

this family, there is a stretch of amino acid in the

ectodomain of the protein core with similarity to the

Cys-rich domain of Frizzled proteins. There are two

unique features in the structural organization of all

glypicans, with potentially important functional

implications.

First and in contrast to syndecans, the attachment

of the GAG chains – mostly HS chains – is located

near the juxtamembrane region. This allows the

three linear HS chains to span a great deal of plasma

membrane surface, thereby presenting various

morphogens and growth factors in an active configuration

to their cognate receptors. Indeed, glypicans

bind to and modulate the activity of Hedgehog (Hh),

Wnt, and FGFs [82–84] . More recently, it has been

shown that glypican-3 binds to Frizzled thereby

acting directly in the modulation of canonical Wnt

signaling [85] .

Second, glypicans are dually processed via partial

proteases and lipases. In the former case, the

ectodomain of glypicans is processed via endoproteolytic

cleavage by a furin-like convertase. This

processing generates two subunits that are then

bound via disulfide bonds, in a way similar to the Met

receptor. In the latter case, the entire glypican

proteoglycan is released from the plasma membrane

via an extracellular lipase (Notum ) that cleaves the

GPI anchor. Drosophila studies have shown that the

Notum -mediated release of glypican can regulate

morphogen gradients including Wnt, BMP and Hh

gradients [84] .

Notably, the anchorless GPC-1, devoid of the

GPI anchor, is a stable -helical protein that rests

high concentrations of urea and guanidine HCL

[86] . Unfolding data are consistent with a two-state

model, suggesting that GPC-1 protein core is

a densely-packed globular protein. In agreement

with these data, the crystal structure of the

Drosophila glypican Dally-like protein has revealed

an extended -helical fold [87] . The

crystal structure of human GPC-1 is very similar

to Drosophila Dally-like , and consists of a stable

-helical domain with 14 conserved Cys residues,

followed by a GAG attachment site that is

exclusively substituted with HS chains [88] . Of

interest, removal of the -helical domain leads to

substitution with CS chains instead of HS chains,

indicating that there is a “ message” embedded in

the -helical domain that drives a different posttranslational

modification [88] .

Functionally, glypicans have been involved in the

control of tumor growth and angiogenesis. For

example, glypican-3 has been implicated in cancer

and growth control. Human mutations of GPC3

cause the rare X-linked Sympson–Golabi–Behmel

(SGB) syndrome, characterized by both pre- and

post-natal overgrowth, abnormal craniofacial features,

cardiovascular anomalies, renal dysplasia and

urinary tract malformations [84] . Originally, it was

hypothesized that GPC3 was an inhibitor of IGF-II,

given the prominent function of IGF-II in developmental

growth. However, it was later found that the

levels of IGF-II do not change in Gpc3/ mice nor

does GPC3 interacts with IGF-II. It appears that

GPC3 is an inhibitor of the Hh signaling, insofar as

the Hh-dependent signaling activity is elevated in

Gpc3/ mice. Moreover, purified glypican-3 binds

with high affinity to Indian and Sonic Hh as well as it

competes with Patched for Hh binding [83,89] . A

recent study has shown that processing by convertases

is required for GPC3-evoked suppression

of Hh signaling, and this process is dependent on the

HS chains and their degree of sulfation [90] . Thus,

the glypican family is not only complex in nature, but

is also the control of various modifying enzymes

(proteases and lipases) that modulate its biological

activity. We are positive than many “ surprises” will

happen in the future regarding unsuspected biological

functions of various glypicans.

Pericellular and basement membrane zone

Proteoglycans.

This group of four proteoglycans is closely

associated with the surface of many cell types

The key for the various modules is

provided in the bottom panel.

anchored via integrins or other receptors, but they

can also be a part of most basement membranes.

Pericellular proteoglycans are mostly HSPGs and

include perlecan and agrin, which share homology

especially at their C-termini, and collagens XVIII and

XV, which share homology at their N- and C-terminal

noncollagenous domains (Fig. 1 ).

Perlecan

Perlecan is a modular HSPG encoded by a large

gene [91,92] with a complex promoter [93–95] . The

~500-kDa protein core is composed of 5 domains

with homology to SEA, N-CAM, IgG, LDL receptor

and laminin [96,97] (Fig. 3 ). The terminal LG3

domain has been crystallized and reveals a jellyroll

fold characteristic of other LG modules [3] . Perlecan

is expressed by both vascular and avascular tissues

[97–101] , and is ubiquitously located at the apical

cell surface [102,103] and basement membranes

[98,104–106] . Perlecan regulates various biological

processes primarily because of its widespread

distribution [101,105] and its ability to interact with

various ligands and RTKs [107] , and more recently

the potential utilization of perlecan splice variants in

mast cells [108] . Perlecan is an early responsive

gene and is induced by TGF [109] and repressed

by interferon [95] . The heparan sulfate chains

of perlecan and the protein core can be cleaved by

heparanase and various proteases [110–112] ,

respectively, releasing various pro-angiogenic

Perlecan is involved in modulating cell adhesion

[114,115] , lipid metabolism [116] , thrombosis and cell

death [117,118] , biomechanics of blood vessels and

cartilage [119–121] , skin and endochondral bone

formation [122,123] , and osteophyte formation [124] .

Perlecan binds and modulates the activity of several

growth factors and morphogens [106,125–129] and

its expression is often deregulated in several types of

cancer [130–134] . In Drosophila , perlecan, known as

Trol (for terribly reduced optical lobe) regulates Fgf

and Hh signaling to activate neural stem signaling

[135,136] . In addition, Trol is essential for the

architecture and maintenance of the lymph gland

and for the proliferation of blood progenitor cells [137] .

Loss of Trol is associated with premature differentiation

of hemocytes and this phenotype can be rescued

by ectopic expression of Hh [137] . In mice, Hspg2

controls neurogenesis in the developing telencephalon

[138] . Moreover, perlecan can act as a lipoprotein

receptor and mediate its endocytosis and catabolism

[116] . Specifically, domain II of perlecan has been

shown to bind low density lipoproteins and this

interaction is mediated by the O-linked oligosaccharides

[139] , suggesting an important role for perlecan

in atherogenesis and lipid retention.

Perlecan is a complex regulator of vascular

biology and tumor angiogenesis [33,140,141] by

performing a dual function: via the N-terminal HS

chains, perlecan is pro-angiogenic [96] by binding

and presenting VEGFA and various FGFs to their

cognate receptors [33,141–152] . Moreover, heparanase-

mediated cleavage of basement membrane

perlecan releases FGF10 and enhances salivary

gland branching morphogenesis [153] . Indeed,

ablating Hspg2 or preventing Hspg2 expression in

early embryogenesis causes severe cardiovascular

defects [154–157] . The critical role for the N-terminal

HS chains of perlecan has been elegantly demonstrated

by the generation of mice harboring a

genomic deletion of exon 3, designated Hspg 23/3

mice, which encodes the SGDs responsible for the

covalent attachment of HS chains [158] . These

mutant mice have impaired angiogenesis, delayed

healing after experimental wounding and suppression

of tumor growth [159] . When challenged with

flow cessation of the carotid artery, the Hspg 23/3

mice show an enhanced intimal hyperplasia and

smooth muscle cell proliferation [160,161] . Moreover,

during mouse hind-limb ischemia, the HS chains

of perlecan are key regulators of the angiogenic

response [162] .Collectively, these studies reaffirmthe

role of HS perlecan in modulating pro-angiogenic

factors such as FGF2, VEGFA and PDGF.

More recently other functions of perlecan have

been discovered. Using a lethality-rescued Hspg2/

where perlecan was reintroduced into the cartilage, it

was found that perlecan deficiency leads to significant

depression of endothelial nitric oxide synthase

[163] . This leads to endothelial cell dysfunction, as

shown by attenuated endothelial relaxation, likely as

a consequence of endothelial nitric oxide synthase

expression. This is another example of how a

secreted HSPG affects the biology of vascular

endothelial cells likely through a receptor-mediated

signaling pathway. Another recently unveiled function

of perlecan is its ability to bind the clustering

molecule gliomedin [164] . In this case, perlecan

binds dystroglycan at nodes of Ranvier which are

required for fast conduction and accumulation of Na+

channels. Perlecan seems to enhance clustering of

nodes of Ranvier components via a specific interaction

with gliomedin. Thus, perlecan may have

specific roles in the biology and pathophysiology of

peripheral nodes [164] .

In contrast to the pro-angiogenic N-terminal

domain I, the C-terminal processed form of perlecan

domain V, named endorepellin [165] , has a nearly

opposite function: it inhibits endothelial cell migration,

capillary morphogenesis, and in vivo angiogenesis

[166–169] . A global proteomic analysis of

human serum has identified endorepellin as a

major circulating protein [170] . Moreover, endorepellin

has been detected in extracts of fetal cartilage,

exclusively in the hypertrophic zone, and it was

speculated that processing of perlecan protein core

in the growth plate could play a role in inhibiting

blood vessel invasion or formation in cartilage [171] .

Moreover, MMP-7 processing of

perlecan in the prostate cancer stroma acts as a

molecular switch to favor cancer invasion [112] .

Thus, processed forms of perlecan protein core

harboring domains III and IV can function as

protumorigenic factors.

Endorepellin binds to the 2 1 integrin receptor

[140,166,194] , and tumor xenografts generated in

21/ mice are insensitive to systemic delivery of

endorepellin [168] . Endorepellin triggers the activation

of the tyrosine phosphatase SHP-1 which, in

turn, dephosphorylates and inactivates various

RTKs including VEGFR2 [195] . Soluble endorepellin

alters the proteomic profile of human endothelial

cells [196] , and exerts a dual receptor antagonism by

concurrently targeting VEGFR2 and the 2 1

integrin [197] . Notably, the proximal LG1/2 domains

bind the Ig3– 5 domain of VEGFR2 while the terminal

LG3 domain, release by BMP-1/Tolloid-like metalloproteinases

[174] , binds the 2 1 integrin [198] . This

dual signaling causes: (a) Disassembly of actin

filaments and focal adhesions, via the 2 1 integrin,

leading to suppression of endothelial cell migration

[198,199] , and (b) Activation of SHP-1 dephosphorylates

Tyr1175 , a key residue in the cytoplasmic tail of

VEGFR2, and consequent transcriptional inhibition

of VEGFA [200] .

More recently, we have discovered that endorepellin

induces autophagy in endothelial cells via

VEGFR2 signaling [201] , similar to decorin (see

below). This novel function could contribute to the

angiostatic properties of this interesting fragment of

perlecan protein core.

Agrin

The second pericellular/basement membrane

HSPG is agrin. A C-terminal portion of agrin lacking

HS chains was first isolated from the Torpedo

electric organ as an agent responsible for acetylcholine

receptor (AChR) clustering, thereby the

eponym agrin, from the Greek ageirein , meaning

“ to assemble” [202] . The majority of the research on

agrin in mammalians has focused on agrin's

contribution to the control of the postsynaptic

apparatus in the neuromuscular junction. However,

after many years of research, it was serendipitously

discovered that agrin was indeed anHSPGinteracting

with N-CAM in the avian brain [203] . Subsequently,

orthologs of agrin have been cloned from multiple

species and are all highly homologous.

Agrin has a multimodular structural organization

that is homologous to that of perlecan with potential

generation of several splice isoforms.

In addition to secreted full-length brevican, an

isoform of brevican encoded by a shorter 3.3 kb

mRNA and highly expressed during post-natal

development, is linked to the plasma membrane

via a GPI anchor [273] . Notably, the GPI-anchored

brevican lacks EGF, C-type lectin and CRP modules

but contains a stretch of hydrophobic amino acids

resembling the GPI-anchor. Brevican is located at

the outer surface of neurons and is enriched at

perisynaptic sites. Brevican interacts with tenascin-

R and fibulin-2 via its G3-like domain [274] .

Functionally, brevican has been implicated in glioma

tumorigenesis, nervous tissue injury and repair, and in

Alzheimer's disease [274] . However, many more

studies need to be performed before a clear picture

of brevican's biology can be clearly drawn.

Final considerations

Of the 43 genes encoding full-time proteoglycans,

only 33 appear to be glycanated. Thus, roughly 1 in

10,000 genes in the human genome codes for a

proteoglycan protein core. This is quite amazing and

indicates that proteoglycans play fundamental and

often vital functions necessary for life to operate and

evolve.We are confident that new proteoglycans will

be discovered in the future. One of the major

difficulties in finding new proteoglycans is their

large size and negative charge. Both hinder proper

separation in conventional acrylamide or 2D gels

used for routine proteomic studies of various

biological fluids and tissues. However, as in the

case of agrin and collagen XVIII which were studied

for several years without knowing their proteoglycan

nature, it is likely that there will be significant

discoveries of known proteins as being members

of the “restricted” proteoglycan gene family. We

hope that this nomenclature will help researchers

who want to familiarize themselves with our exciting

and growing field of proteoglycan biology.

Explanation / Answer

A protein that are heavily glycosylated. The basic proteoglycan unit consist of a core protein with one or more covalently attached glycosoaminoglycan chain. They are found in all connective tissue. Intracellular proteoglycan , serglycin serve as biological glue for most of intracellular proteses stored within the granule. It is packaged in the granule of most cells. Serglycin bind and modulate activity of several inflammatory mediator chemokines, cytokines and growth factor. It is also found in wide variety of non immune cells such as endothelial, chondrocyte and smooth muscle cells. Cell surface seroglycin promote adhesion of myeloma cells to collagen and affect expressiin of MMP. It is a key componant of cell inflammatory response in activated primary human endothelial cells. It act as marker of immune myeloid cells and interact with TNF- alpha and proteases. It protect breast cancer cells from complement attack and support cancer cell survival. Heparin sulfate proteoglycan (HSPG), syndecan are prevalently associated with cell surface or the pericellular matrix. It is associated with plasma membrane of the cell either directly or via GPI anchor and function as biological modifier of growth factor FGF, VEGF and PDGF. It is responsible for presentation of growth factor to their coagnate receptor in their biological favorable form. It is also participate in long range maintenance of gradient for morphagen during embryogenesis and regenerative processes. Syndecan act as endocytic receptor and involving uptake of exosomes. Soluble syndecan -1 promote the growth of myeloma. Chondrin and dermatan sulfate containing proteoglycan function as structural constituents of complex matrix such as cartilage, brain ,tendons and corneas. They also provide vesoelastic property, retain water, keep osmotic pressure, dictate proper collagen organization. CSPG act as receptor for clostridium difficile toxin B.

Largest class of proteoglycan called small leucin rich proteoglycan (SLRP) function as both structural constituents and signaling molecule. SLRP interact with receptor tyrosine kinase that regulate fundamental process such as migration, proliferation, and angiogenesis.

This review provide functional aspect of proteoglycan.

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