Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

(a) Gram-positive bacteria

Polysaccharide

Peptidoglycan layers (cell wall)

(b) Gram-negative bacteria

Lipopoly- saccharide

Outer lipid bilayer membrane

Cell wall	Peptidoglycan
	Inner lipid	Lipoprotein
	bilayer membrane

FIGURE 9.23 ● 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

)

)

Mannose

Abequose

Rhamnose

D-Galactose

Heptose

KDO

NAG

)

)

O antigen

Core oligosaccharide

O O

P P P

P P P

Protein

peptidoglycan. Diaminopimelic acid replaces one of the D-alanine residues in about 10% of the peptides of the peptidoglycan. On the other end of the hydrophobic protein, the N-terminal residue, a serine, makes a covalent bond to a lipid that is part of the outer membrane.

As shown in Figure 9.24, 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 9.24 ● Lipopolysaccharide (LPS) coats the outer membrane of Gram-negative	▲
bacteria. The lipid portion of the LPS is embedded in the outer membrane and is linked
to a complex polysaccharide.

Lipopolysaccharides

Outer cell wall

Peptidoglycan

Plasma membrane

Proteins

281

FIGURE 9.25

282 Chapter 9 ● Membranes and Cell Surfaces

● Teichoic acids are covalently linked to the peptidoglycan of Gram-

positive bacteria. These polymers of (a, b) glycerol phosphate or (c) ribitol phosphate are linked by phosphodiester bonds.

(Figure 9.24). These many different unique units determine the antigenicity of the bacteria; that is, animal immune systems recognize them as foreign substances and raise antibodies against them. As a group, these antigenic determinants are called the O antigens, 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 Gram-nega- tive cells. Having no outer membrane, Gram-positive cells compensate with a thicker wall. Covalently attached to the peptidoglycan layer are teichoic acids, which often account for 50% of the dry weight of the cell wall (Figure 9.25). The teichoic acids are polymers of ribitol phosphate or glycerol phosphate linked by phosphodiester bonds. In these heteropolysaccharides, the free hydroxyl groups of the ribitol or glycerol are often substituted by glycosidically linked monosaccharides (often glucose or N-acetylglucosamine) or disaccharides. D-Alanine is sometimes found in ester linkage to the saccharides. Teichoic acids are not confined to the cell wall itself, and they may be present in the inner membranes of these bacteria. Many teichoic acids are antigenic, and they also serve as the receptors for bacteriophages in some cases.

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, constitution, and function. Although each animal cell contains, in its genetic material, the instructions to replicate the entire organism, each differentiated

O–

H H H

O–

H H H

O–

H H H

HO

P

O

H2C

C

C

C

CH2

O

P

O

H2C

C

C

C

CH2

O

P

O

H2C

C

C

C

CH2OH

O

O O O

O

O O O

O

O O O

CH2OH

H– or D-Alanine

H– or D-Alanine

Glucose

Glucose

H

O

H or C

O

H

+

OH

H

CHNH

HO

H

3

H

OH

CH3

7

Ribitol teichoic acid from Bacillus subtilis

(a)

(b)

(c)

O

CH2

O

CH2

O

CH2

O

D-Alanine

O

O

Glucose

O

O

CH

CH

P

CH

P

D-Alanine

H2C

O

H2C

O

O

CH

O–

O–

H– or D-Alanine

O

O

CH

P

H2C

O

O–

9.3 ● Membranes and Cell-Surface Polysaccharides

283

animal cell carefully 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 removed from the culture and separated, they lose their synchronous behavior, but if allowed to reestablish cell-to-cell contact, they spontaneously restore their synchronous contractions.

H U M A N B I O C H E M I S T R Y

Selectins, Rolling Leukocytes, and the Inflammatory Response

Human bodies are constantly exposed to a plethora of bacteria, viruses, and other inflammatory substances. To combat these infectious and toxic agents, the body has developed a carefully regulated inflammatory response system. Part of that response is the orderly migration of leukocytes to sites of inflammation. Leukocytes literally roll along the vascular wall and into the tissue site of inflammation. This rolling movement is mediated by reversible adhesive interactions between the leukocytes and the vascular surface.

These interactions involve adhesion proteins called selectins, which are found both on the rolling leukocytes and on the endothelial cells of the vascular walls. Selectins have a characteristic domain structure, consisting of an N-terminal extracellular lectin domain, a single epidermal growth factor (EGR) domain, a series of two to nine short consensus repeat (SCR) domains, a single transmembrane segment, and a short cytoplasmic domain. Lectin domains, first characterized in plants, bind carbohydrates

L-Selectin

Selectin

receptors Leukocyte

Selectin receptor

E-Selectin

Endothelial cell

P-Selectin

with high affinity and specificity. Selectins of three types are known—E-selectins, L-selectins, and P-selectins. L-selectin is found on the surfaces of leukocytes, including neutrophils and lymphocytes, and binds to carbohydrate ligands on endothelial cells. The presence of L-selectin is a necessary component of leukocyte rolling. P-selectin and E-selectin are located on the vascular endothelium and bind with carbohydrate ligands on leukocytes. Typical neutrophil cells possess 10,000 to 20,000 P-selectin binding sites. Selectins are expressed on the surfaces of their respective cells by exposure to inflammatory signal molecules, such as histamine, hydrogen peroxide, and bacterial endotoxins. P-selectins, for example, are stored in intracellular granules and are transported to the cell membrane within seconds to minutes of exposure to a triggering agent.

Substantial evidence supports the hypothesis that selectin– carbohydrate ligand interactions modulate the rolling of leukocytes along the vascular wall. Studies with L-selectin–deficient and P-selectin–deficient leukocytes show that L-selectins mediate weaker adherence of the leukocyte to the vascular wall and promote faster rolling along the wall. P-selectins conversely promote stronger adherence and slower rolling. Thus, leukocyte rolling velocity in the inflammatory response could be modulated by variable exposure of P-selectins and L-selectins at the surfaces of endothelial cells and leukocytes, respectively.

SCR repeat

P-Selectin SS LEC E

SCR repeat

E-Selectin SS LEC E

SCR repeat

L-Selectin SS LEC E

A diagram showing the interactions of selectins with their receptors.

The selectin family of adhesion proteins.

284 Chapter 9 ● Membranes and Cell Surfaces

Kidney cells grown in culture with liver cells seek out and make contact with other kidney cells and avoid contact with liver cells. Cells grown in culture grow freely until they make contact with one another, at which point growth stops, a phenomenon well known as contact inhibition. One important characteristic of cancerous cells is the loss of contact inhibition.

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 carbohydrate 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 can be 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.

9.4 ● Glycoproteins

Many proteins found in nature are glycoproteins because they contain covalently linked oligoand 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 9.26a) or via the amide nitrogen of an asparagine residue (in N-linked saccharides) (Figure 9.26b). The carbohydrate residue linked to the protein in O-linked saccharides is usually an N-acetylgalactosamine, but mannose, galactose, and xylose residues linked to protein hydroxyls are also found (Figure 9.26a). Oligosaccharides O-linked to glycophorin (see Figure 9.14) involve N- acetylgalactosamine linkages and are rich in sialic acid residues (Figure 9.14). N-linked saccharides always have a unique core structure composed of two N- acetylglucosamine residues linked to a branched mannose triad (Figure 9.26b, 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 so that an individual mucin molecule (Mr 107) may extend over a distance of 150 to 200 nm in solution. Inherent 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.

FIGURE 9.26

9.4 ● Glycoproteins

285

(a)—O-linked saccharides

CH2OH

CH2OH

HO

O

HO

O

H

O

H

H

C

O

OH

H

H

H

H

H

O

CH2

C

H Ser

H

OH

H NHCCH3

NH

O

β -Galactosyl–1,3–α -N-acetylgalactosyl-serine

● The carbohydrate moieties of glycoproteins 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 mannose, complex, and hybrid, the latter of which combines structures found in the high mannose and complex saccharides.

HOCH2

CH2OH

O

CH3

C

O

H

H

O

H

C

O

OH

O

CH

C

H

Thr

HO

OH HO

O

CH2

C

H

Ser

OH

NH

H

H

NH

α -Xylosyl-threonine

α

-Mannosyl-serine

(b)—Core oligosaccharides in N-linked glycoproteins

HOCH2

O

OH HO

α

1,6

HO

O

HOCH2

Man

CH2

HOCH2

HOCH2

O

C

O

O

O

O

O

NH

C

CH2

C

H Asn

OH HO

HO

O

OH

O

OH

N

H

β

HO

HO

β 1,4

1,4

O

HN

C

CH3

HN

C

CH3

α 1,3

Man

Man

GlcNAc

GlcNAc

O

O

(c)

N-linked glycoproteins

Man

Man

Man

Sia

Sia

Gal

α

1,2

α 1,2

α 1,2

α

2,3 or 6

α

2,3 or 6

β

1,4

Man

Man

Man

Gal

Gal

GlcNAc

α

1,2

α 1,3

α

1,6

β 1,4

β

1,4

Man

Man

GlcNAc

GlcNAc

β

1,2

Man

Man

α

1,3

α

1,6

β 1,2

β

1,2

α

1,3

α

1,6

Man

Man

Man

Man

Man

β

1,4

α 1,3

α 1,6

α 1,3

α 1,6

GlcNAc

Man

Man

β

1,4

β

1,4

β

1,4

GlcNAc

GlcNAc

GlcNAc

β

1,4

β

1,4

Asn

GlcNAc

GlcNAc

Asn

Asn

286 Chapter 9 ● Membranes and Cell Surfaces

Leukosialin	Decay-accelerating	LDL
	factor (DAF)	receptor

O-linked	Globular
saccharides	protein heads

Glycocalyx

(10 nm)

Plasma membrane

FIGURE 9.27 ● 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, Trends in Biochemical Sciences

15:291–294.)

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 9.27). Leukosialin, like mucin, adopts a highly extended conformation, allowing it to project great distances above the membrane surface, perhaps protecting the cell from unwanted interactions with macromolecules or other cells. The second structural motif is exemplified by the low density lipoprotein (LDL) receptor and by decay accelerating factor (DAF). These proteins contain a highly O-glycosylated stem region that separates the transmembrane domain from the globular, functional extracellular domain. The O-glycosylated stem serves to raise the functional domain of the protein far enough above the membrane surface to make it accessible to the extracellular macromolecules with which it interacts.

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 glycosylated with the disaccharide -galactosyl-(1n3)- -N-acetylgalactos-

9.4 ● Glycoproteins

287

...

N

H

Ala

H3C

C

H

C

O

H

N

H

C

CH3

Ala

HOCH2

O

O

C

HOCH2

HO

N

H

CH

C

H

HO

O

O

NH

O

Thr

C

O

OH

CH3

...

C

O

OH

CH3

β -Galactosyl–1,3–α -N-acetylgalactosamine

Repeating unit of antifreeze glycoproteins

FIGURE 9.28 ● The structure of the repeating unit of antifreeze glycoproteins, a disaccharide consisting of -galactosyl-(1n3)- -N-acetylgalactosamine in glycosidic linkage to a threonine residue.

amine (Figure 9.28). 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, inhibiting further growth of the crystals.

N-Linked Oligosaccharides

N-Linked oligosaccharides are found in many different proteins, including immunoglobulins G and M, ribonuclease B, ovalbumin, and peptide hormones (Figure 9.29). Many different functions are known or suspected for N-glycosy- lation 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 conformations and protect proteins against proteolysis. Eukaryotic organisms use posttranslational additions of N-linked oligosaccharides to direct selected proteins to various intracellular organelles.

Oligosaccharide Cleavage as a Timing Device for Protein Degradation

The slow cleavage of monosaccharide residues from N-linked glycoproteins circulating in the blood targets these proteins for degradation by the organism. The liver contains specific receptor proteins that recognize and bind glycoproteins that are ready to be degraded and recycled. Newly synthesized serum glycoproteins contain N-linked triantennary (three-chain) oligosaccharides having structures similar to those in Figure 9.30, 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

288 Chapter 9 ● Membranes and Cell Surfaces

Ribonuclease B	Human IgG
Man	Sia		Sia
Man	Gal		Gal
Man	GlcNAc		GlcNAc
Man	Man		Man
Man		Man
Man		GlcNAc
GlcNAc		GlcNAc	Fuc
GlcNAc		Asn
Asn
One of several from
	ovalbumin
Gal
GlcNAc GlcNAc		Man	Man
Man	GlcNAc	Man

Man

GlcNAc

GlcNAc

Mannose-6-P groups in certain lysosomal enzymes

Man

Man Man

Man

Man Man

Man Man

Man

GlcNAc

GlcNAc

Asn

Various serum glycoproteins

	NeuNAc	NeuNAc
Gal	Gal	Gal
GlcNAc	GlcNAc	GlcNAc

Man Man

Man

GlcNAc

Sulfated oligosaccharide from bovine luteinizing hormone

Sia

Sia

GlcNAc GalNAc

Man Man

Man

GlcNAc

GlcNAc L-Fuc

Asn

Porcine thyroglobulin

Soybean agglutinin

Man	Man	Man
Man	Man	Man
Man		Man
	Man

GlcNAc

GlcNAc

GlcNAc

L-Fuc

Asn

Asn

Asn

FIGURE 9.29 ● Some of the oligosaccharides found in N-linked glycoproteins.

the liver, the asialoglycoprotein receptor binds the exposed galactose residues of these glycoproteins with very high affinity (KD 10 9 to 10 8 M). 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 galactose 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.

9.5 ● Proteoglycans

289

Sia			Gal	GlcNAc		Man
Sia			Gal	GlcNAc		Man
	Sia		Gal		GlcNAc

(Does not bind)

...

Man GlcNAc GlcNAc Asn

...

Sialic acid

			Gal	GlcNAc		Man
Sia			Gal	GlcNAc		Man
	Sia		Gal		GlcNAc

(Binds poorly)

...

Man GlcNAc GlcNAc Asn

...

Sialic acid

	Gal	GlcNAc		Man
	Gal	GlcNAc		Man
Sia	Gal		GlcNAc

(Binds moderately)

...

Man GlcNAc GlcNAc Asn

...

Sialic acid

Gal GlcNAc Man

...

Man GlcNAc GlcNAc Asn

Gal	GlcNAc Man
Gal		GlcNAc

(Binds tightly to liver asialoglycoprotein receptor)

FIGURE 9.30 ● Progressive cleavage of sialic acid residues exposes galactose residues. Binding to the asialoglycoprotein receptor in the liver becomes progressively more likely as more Gal residues are exposed.

...

9.5 ● Proteoglycans

Proteoglycans are a family of glycoproteins whose carbohydrate moieties are predominantly glycosaminoglycans. The structures of only a few proteoglycans are known, and even these few display considerable diversity (Figure 9.31). They 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 (Table 9.2). 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 composed 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 connective tissues, contains only three Ser-Gly pairs, only one of which is normally glycosylated. In addition to glycosaminoglycan units, proteoglycans may also contain other N-linked and O-linked oligosaccharide groups.

Functions of Proteoglycans

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 integral transmembrane proteins, such as syndecan. Both types of proteoglycan

290	Chapter 9	● Membranes and Cell Surfaces
(a) Versican		NH3+	(b) Serglycin
				NH3+
		Hyaluronic acid
		binding domain
		(link-protein like)
			Ser/Gly	COO– Chondroitin
			protein core	sulfate

Chondroitin sulfate

(c) Decorin

NH+3

Chondroitin/dermatan sulfate chain

Protein core

COO–

(d) Syndecan

Heparan sulfate

NH+3

Extracellular	Chondroitin
domain	sulfate

(e) Rat cartilage proteoglycan

Chondroitin sulfate

O–linked

oligosaccharides

COO–	Cytoplasmic	Keratan sulfate
	domain

Transmembrane domain

Epidermal growth factor-like domains

N–linked

oligosaccharides

COO–

FIGURE 9.31 ● The known proteoglycans include a variety of structures. The carbohydrate groups of proteoglycans are predominantly glycosaminoglycans O-linked to serine residues. Proteoglycans include both soluble proteins and integral transmembrane proteins.

appear to function by interacting with a variety of other molecules through their glycosaminoglycan components and through specific receptor domains in the polypeptide itself. For example, syndecan (from the Greek syndein meaning “to bind together”) is a transmembrane proteoglycan that associates intracellularly with the actin cytoskeleton (Chapter 17). Outside the cell, it interacts with fibronectin, an extracellular 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 extracellular space, linking components of the extracellular matrix, facilitating the binding of cells to the matrix, and mediating the binding of growth factors and other soluble molecules to the matrix and to cell surfaces (Figure 9.32).