Biochemistry, 4th Edition P25 pot

Cell Walls of Gram-Negative Bacteria In Gram-negative bacteria, the peptidogly-can wall is the rigid framework around which is built an elaborate membrane struc-ture Figure 7.30.. As a g

Cell Walls of Gram-Negative Bacteria In Gram-negative bacteria, the

peptidogly-can wall is the rigid framework around which is built an elaborate membrane

struc-ture (Figure 7.30) The peptidoglycan layer encloses the periplasmic space and is

at-tached to the outer membrane via a group of hydrophobic proteins.

As shown in Figure 7.31, the outer membrane of Gram-negative bacteria is

coated with a highly complex lipopolysaccharide, which consists of a lipid group

(anchored in the outer membrane) joined to a polysaccharide made up of long

chains with many different and characteristic repeating structures (Figure 7.31)

These many different unique units determine the antigenicity of the bacteria; that

is, animal immune systems recognize them as foreign substances and raise

antibod-ies against them As a group, these antigenic determinants are called the O antigens,

Gram-positive bacteria

Polysaccharide coat

Peptidoglycan layers (cell wall)

Lipopoly-saccharide

Cell wall

Outer lipid

bilayer membrane

Peptidoglycan

Inner lipid

bilayer membrane

(a)

Gram-negative bacteria

(b)

FIGURE 7.30 The structures of the cell wall and membrane(s) in Gram-positive and Gram-negative bacteria.

The Gram-positive cell wall is thicker than that in Gram-negative bacteria, compensating for the absence of a

second (outer) bilayer membrane.

Lipopolysaccharide

D -Galactose

Mannose

Rhamnose

Heptose

KDO NAG

Core oligo-saccharide

O antigen

Proteins

Plasma membrane Peptidoglycan Outer cell wall Lipopolysaccharides

Abequose

Protein

P P P

P P

P

䊳 FIGURE 7.31 Lipopolysaccharide (LPS) coats the outer membrane of Gram-negative bacteria The lipid

por-tion of the LPS is embedded in the outer membrane and is linked to a complex polysaccharide.

and there are thousands of different ones The Salmonella bacteria alone have well

over a thousand known O antigens that have been organized into 17 different groups The great variation in these O antigen structures apparently plays a role in the recognition of one type of cell by another and in evasion of the host immune system

Cell Walls of Gram-Positive Bacteria In Gram-positive bacteria, the cell exterior

is less complex than for negative cells Having no outer membrane, Gram-positive cells compensate with a thicker wall Covalently attached to the

peptido-glycan layer are teichoic acids, which often account for 50% of the dry weight of

the cell wall The teichoic acids are polymers of ribitol phosphate or glycerol phosphate

linked by phosphodiester bonds

Animals Display a Variety of Cell Surface Polysaccharides

Compared to bacterial cells, which are identical within a given cell type (except for

O antigen variations), animal cells display a wondrous diversity of structure, consti-tution, and function Although each animal cell contains, in its genetic material, the instructions to replicate the entire organism, each differentiated animal cell care-fully controls its composition and behavior within the organism A great part of each cell’s uniqueness begins at the cell surface This surface uniqueness is critical

to each animal cell because cells spend their entire life span in intimate contact with other cells and must therefore communicate with one another That cells are able

to pass information among themselves is evidenced by numerous experiments For

example, heart myocytes, when grown in culture (in glass dishes), establish synchrony

when they make contact, so that they “beat” or contract in unison If they are re-moved from the culture and separated, they lose their synchronous behavior, but if allowed to reestablish cell-to-cell contact, they spontaneously restore their synchro-nous contractions

As these and many other related phenomena show, it is clear that molecular structures on one cell are recognizing and responding to molecules on the

adjacent cell or to molecules in the extracellular matrix, the complex “soup” of

connective proteins and other molecules that exists outside of and among cells

Many of these interactions involve glycoproteins on the cell surface and proteoglycans

in the extracellular matrix The “information” held in these special carbohydrate-containing molecules is not encoded directly in the genes (as with proteins) but is determined instead by expression of the appropriate enzymes that assemble car-bohydrate units in a characteristic way on these molecules Also, by virtue of the several hydroxyl linkages that can be formed with each carbohydrate monomer, these structures are arguably more information-rich than proteins and nucleic acids, which can form only linear polymers A few of these glycoproteins and their unique properties are described in the following sections

Function in Cells?

Many proteins found in nature are glycoproteins because they contain covalently linked oligosaccharide and polysaccharide groups The list of known glycoproteins includes structural proteins, enzymes, membrane receptors, transport proteins, and immunoglobulins, among others In most cases, the precise function of the bound carbohydrate moiety is not understood

Carbohydrate groups may be linked to polypeptide chains via the hydroxyl

groups of serine, threonine, or hydroxylysine residues (in O-linked saccharides) (Figure 7.32a) or via the amide nitrogen of an asparagine residue (in N-linked

saccharides) (Figure 7.32b) The carbohydrate residue linked to the protein in

O

H

H

HO

H

CH2OH

O H H

CH2OH

O

O H

OH

2 C H NH Ser

-Galactosyl-1,3--N-acetylgalactosyl-serine

O

OH

OH

O

O

O HO H HO

H

CH2OH

O

H OH

-Mannosyl-serine

(a) O-linked saccharides

Man

 1,2  1,2

Man

 1,2

Sia

 2,3 or 6

 1,4

 2,3 or 6

Man

 1,2

Man

 1,3

Man

 1,6

 1,4

 1,4

Man

 1,2

 1,2

 1,2

 1,3  1,6

 1,3  1,6

Man

 1,3

Man

 1,6

 1,4

GlcNAc

 1,4

GlcNAc

 1,4

(c) N-linked glycoproteins

CH3

-Xylosyl-threonine

Man

O

C C NH

C NH O

O HO

HOCH2

Man

CH2 O HO HO

Man

O

HO

HO OH

O

O OH

O OH

Man

O

GlcNAc

(b) Core oligosaccharides in N-linked glycoproteins

 1,6

 1,3

O

O

GlcNAc

O C

CH2

O

C

N

O

H

Asn

Man

Man

Man Man

Man

Man

FIGURE 7.32 The carbohydrate moieties of

glycopro-teins may be linked to the protein via (a) serine or

threonine residues (in the O-linked saccharides) or

(b) asparagine residues (in the N-linked saccharides) (c) N-linked glycoproteins are of three types: high

man-nose, complex, and hybrid, the latter of which combines structures found in the high mannose and complex saccharides.

O-linked saccharides is usually an N-acetylgalactosamine, but mannose, galactose, and

xylose residues linked to protein hydroxyls are also found (Figure 7.32a)

Oligosac-charides O-linked to glycophorin (see Figure 9.10) involve N-acetylgalactosamine

linkages and are rich in sialic acid residues N-linked saccharides always have a

unique core structure composed of two N-acetylglucosamine residues linked to a

branched mannose triad (Figure 7.32b, c) Many other sugar units may be linked to each of the mannose residues of this branched core

O-linked saccharides are often found in cell surface glycoproteins and in

mucins,the large glycoproteins that coat and protect mucous membranes in the respiratory and gastrointestinal tracts in the body Certain viral glycoproteins also contain O-linked sugars O-linked saccharides in glycoproteins are often found clustered in richly glycosylated domains of the polypeptide chain Physical studies

on mucins show that they adopt rigid, extended structures An individual mucin molecule (Mr 107) may extend over a distance of 150 to 200 nm in solution In-herent steric interactions between the sugar residues and the protein residues in these cluster regions cause the peptide core to fold into an extended and relatively rigid conformation This interesting effect may be related to the function of O-linked saccharides in glycoproteins It allows aggregates of mucin molecules to form extensive, intertwined networks, even at low concentrations These viscous networks protect the mucosal surface of the respiratory and gastrointestinal tracts from harmful environmental agents

There appear to be two structural motifs for membrane glycoproteins containing

O-linked saccharides Certain glycoproteins, such as leukosialin, are O-glycosylated

throughout much or most of their extracellular domain (Figure 7.33) Leukosialin, like mucin, adopts a highly extended conformation, allowing it to project great dis-tances above the membrane surface, perhaps protecting the cell from unwanted in-teractions with macromolecules or other cells The second structural motif is

ex-emplified by the low-density lipoprotein (LDL) receptor and by decay-accelerating

factor (DAF).These proteins contain a highly O-glycosylated stem region that sep-arates the transmembrane domain from the globular, functional extracellular domain The O-glycosylated stem serves to raise the functional domain of the pro-tein far enough above the membrane surface to make it accessible to the extracel-lular macromolecules with which it interacts

Leukosialin

O-linked saccharides

Decay-accelerating factor (DAF)

LDL receptor

Globular protein heads

Glycocalyx (10 nm)

Plasma membrane

FIGURE 7.33 The O-linked saccharides of glycoproteins

appear in many cases to adopt extended conformations

that serve to extend the functional domains of these

proteins above the membrane surface (Adapted from

Jentoft, N., 1990 Why are proteins O-glycosylated? Trends in

Bio-Polar Fish Depend on Antifreeze Glycoproteins

A unique family of O-linked glycoproteins permits fish to live in the icy seawater of

the Arctic and Antarctic regions, where water temperature may reach as low as

1.9°C Antifreeze glycoproteins (AFGPs) are found in the blood of nearly all

Antarctic fish and at least five Arctic fish These glycoproteins have the peptide

structure

[Ala-Ala-Thr]n-Ala-Ala

where n can be 4, 5, 6, 12, 17, 28, 35, 45, or 50 Each of the threonine residues is

gly-cosylated with the disaccharide -galactosyl-(1⎯ →3)--N-acetylgalactosamine (Figure

7.34) This glycoprotein adopts a flexible rod conformation with regions of threefold

left-handed helix The evidence suggests that antifreeze glycoproteins may inhibit the

formation of ice in the fish by binding specifically to the growth sites of ice crystals,

in-hibiting further growth of the crystals

N-Linked Oligosaccharides Can Affect the Physical Properties

and Functions of a Protein

N-linked oligosaccharides are found in many different proteins, including

immuno-globulins G and M, ribonuclease B, ovalbumin, and peptide hormones Many

differ-ent functions are known or suspected for N-glycosylation of proteins Glycosylation

can affect the physical and chemical properties of proteins, altering solubility, mass,

and electrical charge Carbohydrate moieties have been shown to stabilize protein

con-formations and protect proteins against proteolysis Eukaryotic organisms use

post-translational additions of N-linked oligosaccharides to direct selected proteins to

A DEEPER LOOK

Drug Research Finds a Sweet Spot

A variety of diseases are being successfully treated with sugar-based

therapies As this table shows, several carbohydrate-based drugs are

either on the market or at various stages of clinical trials Some of these drugs are enzymes, whereas others are glycoconjugates

Cerezyme

(imiglucerase)

Vancocin

(vancomycin)

Vevesca

(OGT 918)

GMK

Staphvax

Bimosiamose

(TBC1269)

GCS-100

GD0039

(swainsonine)

PI-88

Adapted from Maeder, T., 2002 Sweet medicines Scientific American 287:40–47.

Additional References: Alper, J., 2001 Searching for medicine’s sweet spot Science 291:2338–2343 Borman, S., 2007 Sugar medicine Chemical & Engineering News 85:19–30.

This enzyme degrades glycolipids, compensating for an enzyme deficiency that causes Gaucher’s disease

A very potent glycopeptide antibiotic that is typically used against antibiotic-resistant infections It inhibits synthesis of peptidoglycan in the bacterial cell wall

A sugar analog that inhibits synthesis of the glycolipid that accumulates in Gaucher’s disease

A vaccine containing ganglioside GM2; it triggers an immune response against cancer cells carrying GM2

A vaccine that is a protein with a linked bacterial sugar; it is intended to treat

Staphylococcus infection.

A sugar analog that inhibits selectin-based inflammation in blood vessels

A sugar that blocks action of a sugar-binding protein on tumors

A sugar analog that inhibits synthesis of carbohydrates essential to tumor metastasis

A sugar that inhibits growth factor–dependent angiogenesis and enzymes that promote metastasis

Genzyme Cambridge, MA Eli Lilly Indianapolis, IN Oxford GlycoSciences Abingdon, UK Progenics Pharmaceuticals Tarrytown, NY

NABI Pharmaceuticals Boca Raton, FL Texas Biotechnology Houston, TX GlycoGenesys Boston GlycoDesign Toronto, Canada Progen

Darra, Australia

various membrane compartments Recent evidence indicates that N-linked oligosac-charides promote the proper folding of newly synthesized polypeptides in the endo-plasmic reticulum (see A Deeper Look on page 209)

Oligosaccharide Cleavage Can Serve as a Timing Device for Protein Degradation

The slow cleavage of monosaccharide residues from N-linked glycoproteins circu-lating in the blood targets these proteins for degradation by the organism The liver contains specific receptor proteins that recognize and bind glycoproteins that are

HO

HOCH2 O OH OH

O HO

O O NH C

CH3 O

CH3

C N

C N

CH3 H

H

H

H3C

H

Ala

Ala

Thr

-Galactosyl-1,3--N-acetylgalactosamine

Repeating unit of antifreeze glycoproteins

O C

C O

O C

FIGURE 7.34 The structure of the repeating unit of

antifreeze glycoproteins, a disaccharide consisting of

-galactosyl-(1⎯ →3)--N-acetylgalactosamine in

glycosidic linkage to a threonine residue.

(Does not bind)

(Binds tightly to liver asialoglycoprotein receptor)

Sia Sia

(Binds poorly)

Sia Sia

(Binds moderately)

Sia

Sialic acid

Sialic acid

Sialic acid

FIGURE 7.35 Progressive cleavage of sialic acid residues

exposes galactose residues Binding to the

asialoglyco-protein receptor in the liver becomes progressively

more likely as more Gal residues are exposed.

ready to be degraded and recycled Newly synthesized serum glycoproteins contain

N-linked triantennary (three-chain) oligosaccharides having structures similar to

those in Figure 7.35, in which sialic acid residues cap galactose residues As these

glycoproteins circulate, enzymes on the blood vessel walls cleave off the sialic acid

groups, exposing the galactose residues In the liver, the asialoglycoprotein

recep-torbinds the exposed galactose residues of these glycoproteins with very high

affin-ity (KD 109to 108M) The complex of receptor and glycoprotein is then taken

into the cell by endocytosis, and the glycoprotein is degraded in cellular lysosomes.

Highest affinity binding of glycoprotein to the asialoglycoprotein receptor requires

three free galactose residues Oligosaccharides with only one or two exposed

galac-tose residues bind less tightly This is an elegant way for the body to keep track of

how long glycoproteins have been in circulation Over a period of time—anywhere

from a few hours to weeks—the sialic acid groups are cleaved one by one The

longer the glycoprotein circulates and the more sialic acid residues are removed,

the more galactose residues become exposed so that the glycoprotein is eventually

bound to the liver receptor

in Cells and Organisms?

Proteoglycans are a family of glycoproteins whose carbohydrate moieties are

pre-dominantly glycosaminoglycans The structures of only a few proteoglycans are

known, and even these few display considerable diversity (Figure 7.36) Those

known range in size from serglycin, having 104 amino acid residues (10.2 kD), to

versican,having 2409 residues (265 kD) Each of these proteoglycans contains one

or two types of covalently linked glycosaminoglycans In the known proteoglycans,

the glycosaminoglycan units are O-linked to serine residues of Ser-Gly dipeptide

sequences Serglycin is named for a unique central domain of 49 amino acids

com-posed of alternating serine and glycine residues The cartilage matrix proteoglycan

contains 117 Ser-Gly pairs to which chondroitin sulfates attach Decorin, a small

proteoglycan secreted by fibroblasts and found in the extracellular matrix of

con-nective tissues, contains only three Ser-Gly pairs, only one of which is normally

gly-cosylated In addition to glycosaminoglycan units, proteoglycans may also contain

other N-linked and O-linked oligosaccharide groups

Functions of Proteoglycans Involve Binding to Other Proteins

Proteoglycans may be soluble and located in the extracellular matrix, as is the case

for serglycin, versican, and the cartilage matrix proteoglycan, or they may be

inte-gral transmembrane proteins, such as syndecan Both types of proteoglycan appear to

A DEEPER LOOK

N-Linked Oligosaccharides Help Proteins Fold

One important effect of N-linked oligosaccharides in eukaryotic

or-ganisms may be their contribution to the correct folding of certain

globular proteins This adaptation of saccharide function allows

cells to produce and secrete larger and more complex proteins at

high levels Inhibition of glycosylation leads to production of

mis-folded, aggregated proteins that lack function Certain proteins are

highly dependent on glycosylation, whereas others are much less

so, and certain glycosylation sites are more important for protein

folding than are others

Studies with model peptides show that oligosaccharides can al-ter the conformational preferences near the glycosylation sites In addition, the presence of polar saccharides may serve to orient that portion of a peptide toward the surface of protein domains However, it has also been found that saccharides usually are not essential for maintaining the overall folded structure after a gly-coprotein has reached its native, folded structure

Source: Helenius, A., and Aebi, M., 2001 Intracellular functions of N-linked

glycans Science 291:2364–2369.

function by interacting with a variety of other molecules through their glycosami-noglycan components and through specific receptor domains in the polypeptide

it-self For example, syndecan (from the Greek syndein, meaning “to bind together”)

is a transmembrane proteoglycan that associates intracellularly with the actin

cyto-skeleton (see Chapter 16) Outside the cell, it interacts with fibronectin, an

extra-cellular protein that binds to several cell surface proteins and to components of the extracellular matrix The ability of syndecan to participate in multiple interactions with these target molecules allows them to act as a sort of “glue” in the extracellu-lar space, linking components of the extracelluextracellu-lar matrix, facilitating the binding of

(a) Versican

Hyaluronic acid–

binding domain (link-protein-like)

Chondroitin sulfate

Protein core

Epidermal growth factor–like domains

COO–

(b) Serglycin

COO–

Ser/Gly protein core

Chondroitin sulfate

(c) Decorin

COO–

Chondroitin/dermatan sulfate chain

(d) Syndecan

COO–

Heparan sulfate

Chondroitin sulfate

Extracellular domain

Cytoplasmic domain Transmembrane

domain

(e) Rat cartilage proteoglycan

Chondroitin sulfate

O-linked oligosaccharides

Keratan sulfate

N-linked oligosaccharides

NH3+

NH3+

FIGURE 7.36 The known proteoglycans include a variety of structures The carbohydrate groups of proteogly-cans are predominantly glycosaminoglyproteogly-cans O-linked to serine residues Proteoglyproteogly-cans include both soluble proteins and integral transmembrane proteins.

cells to the matrix, and mediating the binding of growth factors and other soluble

molecules to the matrix and to cell surfaces (Figure 7.37)

Many of the functions of proteoglycans involve the binding of specific proteins to

the glycosaminoglycan groups of the proteoglycan The glycosaminoglycan-binding

sites on these specific proteins contain multiple basic amino acid residues The

amino acid sequences BBXB and BBBXXB (where B is a basic amino acid and X

is any amino acid) recur repeatedly in these binding domains Basic amino acids

such as lysine and arginine provide charge neutralization for the negative

charges of glycosaminoglycan residues, and in many cases, the binding of

extra-cellular matrix proteins to glycosaminoglycans is primarily charge-dependent For

example, more highly sulfated glycosaminoglycans bind more tightly to fibronectin

However, certain protein–glycosaminoglycan interactions require a specific

carbo-hydrate sequence A particular pentasaccharide sequence in heparin, for example,

binds tightly to antithrombin III (Figure 7.38), accounting for the anticoagulant

properties of heparin Other glycosaminoglycans interact much more weakly

Proteoglycans May Modulate Cell Growth Processes

Several lines of evidence raise the possibility of modulation or regulation of cell

growth processes by proteoglycans First, heparin and heparan sulfate are known

to inhibit cell proliferation in a process involving internalization of the

gly-cosaminoglycan moiety and its migration to the cell nucleus Second, fibroblast

growth factor binds tightly to heparin and other glycosaminoglycans, and the

(Outside)

Fibronectin

Binding site

Binding site Growth

factor

bound to

heparan

sulfate in

matrix

Growth factor receptor

(Inside)

Cytoskeleton (actin)

Membrane heparan sulfate proteoglycan

Chondroiton sulfate proteoglycan

Extracellular matrix

Integrin receptor for fibronectin

FIGURE 7.37 Proteoglycans serve a variety of functions

on the cytoplasmic and extracellular surfaces of the plasma membrane Many of these functions appear

to involve the binding of specific proteins to the glycosaminoglycan groups.

OH

HNR''

OSO3–

O

OH

O

O

COO–

OR'

OSO3–

HNSO3–

OH OSO3–

OH HNSO3–

– (*)

O

FIGURE 7.38 A portion of the structure of heparin, a car-bohydrate having anticoagulant properties It is used by blood banks to prevent the clotting of blood during do-nation and storage and also by physicians to prevent the formation of life-threatening blood clots in patients re-covering from serious injury or surgery This sulfated pen-tasaccharide sequence in heparin binds with high affin-ity to antithrombin III, accounting for this anticoagulant activity The 3-O-sulfate marked by an asterisk is essential for high-affinity binding of heparin to antithrombin III.

heparin–growth factor complex protects the growth factor from degradative en-zymes, thus enhancing its activity There is evidence that binding of fibroblast growth factors by proteoglycans and glycosaminoglycans in the extracellular

ma-trix creates a reservoir of growth factors for cells to use Third, transforming

growth factor ␤ has been shown to stimulate the synthesis and secretion of

pro-teoglycans in certain cells Fourth, several proteoglycan core proteins, including

versican and lymphocyte homing receptor, have domains similar in sequence to those of epidermal growth factor and complement regulatory factor These

growth factor domains may interact specifically with growth factor receptors in the cell membrane in processes that are not yet understood

Proteoglycan

O Ser

Xyl Gal Gal O GluA O GluNAc O GluA O GluNAc O

O Ser

O Gal O GluNAc O Gal O

O Ser

GalNAc

NeuNAc

N Asn

GlcNAc GlcNAc Man

GlcNAc GlcNAc

NeuNAc NeuNAc GalNAc

Core protein Link protein Hyaluronic acid

Carboxylate group

Core protein

Hyaluronic acid

Link protein

Sulfate group

FIGURE 7.39 Hyaluronate (see Figure 7.28) forms the

backbone of proteoglycan structures, such as those

found in cartilage The proteoglycan subunits consist

of a core protein containing numerous O-linked and

N-linked glycosaminoglycans In cartilage, these highly

hydrated proteoglycan structures are enmeshed in a

network of collagen fibers Release (and subsequent

re-absorption) of water by these structures during

com-pression accounts for the shock-absorbing qualities of

cartilaginous tissue.