Polysaccharides

These contain many monosaccharides in glycosidic links, and may contain many branches. They serve as either structural components or energy storage molecules. The most common polysaccharides consisting of single monosaccharides are:

• starch (found in plants). It's a polymer of Glc linked in a main chain through a 1->4 links with a 1->6 branches.  Amylose is starch with no branches, while amylopectin has branches.  Starch granules consist of about 20% amylose and 80% amylopectin.

• glycogen, the main CHO storage in animals. Muscle and liver glycogen consists of Glc residues in a 1->4 links with lots of a 1->6 branches (many more branches than in starch).   The polymer is synthesized on a protein primer called glycogenin (G), and has a structure shown below (in which only 5 rings of the structure are shown instead of the actual 12. (Meléndez-Hevia et al. ) 

• celluose, a structural Glc polymer in plants, consists of b 1->4 links. It is held together by intra and inter-chain H-bonds. It is the most abundant biological molecule in nature.
• chitin, the major substance in exoskeletons of anthropods and mollusks is a b 1->4 linked polymer of GlcNAc.  

The basic chemical structures of these homopolymers are shown below.

Glc b (1-4) Glc link

It makes great chemical sense to store Glc residues as either glycogen or starch, which is one large molecule. A review of colligative properties would inform you that if all the Glc was stored as the monosaccharide, a great osmotic pressure difference would be found between the outside and inside of the cell.  It makes more sense to have glycogen exist as a many-branched linear polymer. When Glc is needed, it is cleaved one residue at a time from all the branches (at the nonreducing ends), producing a large amount of free Glc in a short time.

 Phi/Psi angles can also be described for the starch/glycogen main chain (around the acetal O) in a fashion comparable to the for proteins (around the alpha carbon).  The phi torsion angle describes rotation around the C1-O bond of the acetal link, while the psi angle describes rotation around the O-C4 bond of the same acetal link, with the glucopyranose ring considers as a rigid rotator (just as the 6 atoms in the planar peptide bond unit).  The most extended form of a Glc_n polymer occurs when the glycosidic link is  B1->4 (as in cellulose), which forms linear chains.  The a 1->4 linked main chain of glycogen and starch causes the chain to turn and form a large helix, into which can fit iodine (or I₃^-), which turns starch purple.

Many polysaccharides consist of repeating dissacharides units. These include the following glycosoaminoglycans (GAGs), all which contain one amino sugar and in which one or both of the sugars contain negatively charged sulfate or carboxyl groups. The extent and position of sulfation varies widely between and within GAGs.

• hyaluronic acid, a polymer of Glucuronate (b 1->3) GlcNAc
• dermatan sulfate, L-iduronate (b 1->3 ) GalNAc-4-sulfate
• keratan sulfate, D-Gal (b 1->4) GlcNAc-6-sulfate
• chondrotin sulfate, D-glucuronate (b 1->3) GalNAc-4 or 6-sulfate
• heparin - D-glucuronate-2-sulfate (a 1->4) GlcNSulfo-6-sulfate

GAGs are found in the vitreous humor of the eye and synovial fluid of joints, and in connective tissue like tendons, cartilage, etc, as well as skin. They are found in the extracellular matrix and are often covalently attached to proteins to form proteolglycans.

COMPLEX OLIGOSACCHARIDE DERIVATIVES

1. Cell Membranes of Bacteria

In contrast to eukaryotic cells, bacteria cells have a cell wall in addition to a lipid bilayer membrane. These are essentially carbohydrate polymers which offer protection from exterior hypotonic condition and the high internal osmotic pressures, preventing swelling and bursting of the cells. The membrane consist of a peptidoglycan. Two types exists.

a. Gram positive bacteria- These bacterial can be stained with Gram stain. The wall consists of a GlcNAc (b 1->4) MurNAc repeat (like that in chitin which is a polymer of GlcNAc in (b 1->4) links, but in which the OH of lactate is in ether-linkage to C3 to form N-Acetylmuramic acid).   A tetrapeptide (Ala-D-isoGlu-Lys-D-Ala) is attached in amide link to the carboxyl group of the lactate in MurNAc. The GlcNAc (b 1->4) MurNAc strands are covalently connected by a pentaglycine bridge through the epsilon amino group of the tetrapeptide Lys on one strand and the D-Ala of a tetrapeptide on another strand.

One final structure is found in Gram + membranes. Techioic acids are often attached to the C6 of MurNAc. Teichoic acid is a polymer of glycerol or ribitol to which alternative GlcNAc and D-Ala are linked to the middle C of the glycerol. Multiple glycerols are linked through phosphodiester bonds. These teichoic acids often make up 50% of the dry weight of the cell wall, and present a foreign (or antigenic) surface to infected hosts. These often serve as receptors for viruses that infect bacteria (called bacteriophages).

b. Gram negative bacteria-

These bacterial can NOT be stained with Gram stain. The wall consists of the same structure as in Gram positive bacteria, but the GlcNAc (b 1->4) MurNAc strands are covalently connected through a direct amide bond between the epsilon amino group of the tetrapeptide Lys on one strand and the D-Ala of a tetrapeptide on another strand. (i.e. no pentaGly spacer). In addition, Gram negative bacterial don't have teichoic acid polymers. Rather they have a second, outer lipid bilayer. The cell wall is sandwiched between the inner and outer bilayers. The space between the lipid bilayers is called the periplasmic space. A hydrophobic protein covalently attaches (through an amide link from a protein Lys) to the cell wall at the last amino acid in the tetrapeptide unit of the wall (actually diaminopimelic acid which replaces about 10% of the D-Ala in the cell wall). The N-terminal of the hydrophobic proteins attaches to the outer lipid membrane  through a Ser.  The outer membrane is coated with a lipopolysaccharide (LPS) of varying composition. The LPS determines the antigenicity of the bacteria. The different LPS are called the O-antigens.

Figure:  A detailed view of LPS

A recent article (Designing surfaces that kill bacteria on contact by Joerg C. Tiller, Chun-Jen Liao, Kim Lewis, and Alexander M. Klibanov, Proc. Natl. Acad. Sci. USA, May 22, 2001) describes a new cationic surface which can kill bacteria on contact.  Abstract:  "Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 ± 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 ± 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood."  This represents a chemical killing similar to bleach in that a single gene or gene target is not targeted by the surface.   The polymer has a permanent positive charge, which interacts with the negative cell membrane and wall, leading to killing of the cells.  A simple mutation would not make the bacteria resistant to the surface since wholesale changes in gene structure would have to occur to make tha bacteria resistant.   

2. Glycoprotens

Many proteins, especially those destined for secretion or insertion into membranes, are post-translationally modified by attachment of carbohydrates. They are usually attached through either Asn or Ser side chains. Carbohydrate modifications on the protein appear to be involved in recognition of other binding molecules, prevention of aggregation during protein folding, protection from proteolysis, and increases half-life of the proteins. In contrast to a protein sequence which is determined by the DNA sequence, sugars are attached to proteins by enzymes which recognize appropriate sites on proteins and attach the sugars. Since there are many sugars which contain many functional groups that can serve as potential attachment sites, the structures of the oligosaccharides attached to proteins are enormously varied, complex, and hence "information rich" compared to linear or folded polymers like DNA and proteins.

a. N-linked Glycoproteins

These contain CHOs attached through either a GlcNAc or GalNAc to an Asn in a X-Asn-X-Thr sequence of the protein. There are three types of N-linked glycoproteins, high mannose, complex, and hybrid.  They all contain the same core oligosaccharide - (Man)₃(GlcNAc)₂ attached to Asn.

High Mannose N-linked Oligosaccharides

Complex N-linked Oligosaccharides

Hybrid N-linked Oligosaccharides

Note that in the hybrid oligosaccharide, one terminus contains Gal(b-1,4)GlcNAc.  However, in all other mammals except man, apes, and old world monkey, an additional Gal is often connected in an a-1,3 link to the Gal to give a terminus of:  Gal(a-1,3)Gal(b-1,4)GlcNAc.  These animals have an additional enzyme, a-1,3 Gal transferase.  Bacteria also have this enzyme and since we have been exposed to this link through bacterial infection, we mount an immune response against it.  Why is this important?  Pig hearts turn out to be similar to human hearts, so they might be good candidates for transplantation into humans (xenotransplants).  However, the Gal-a-1,3-Gal link is recognized as foreign, and we mount a significant immune response against it.  Several biotech firms are trying to delete the pig a-1,3 Gal transferase which would prevent the addition of the terminal Gal, and make them good donors for transplanted hearts.

b. O-linked Glycoproteins

The CHOs are usually attached from a Gal (b 1-3) GalNAc to a Ser or Thr of a protein. The blood group antigens (CHOs on cells attached to either proteins or lipids) are examples .

3. Proteoglycans - PGs

Some proteins are so modified with CHOs that they contain more CHOs than amino acids. Proteins  linked to glycosoaminoglycans are together called proteoglycans. (PGs) The structure of a few proteoglycans is known. The GAGs are O-linked to the protein typically to a Ser of a Ser-Gly dipeptide often repeated in the protein. Some of the proteoglycans also contained N-linked oligosaccharide groups.

PGs can be soluble and found in the extracellular matrix, or as integral membrane proteins. Given the diversity of sugars and the varying extent of sulfation, the CHO part of PGs provide an incredible vary of binding structures to at or near the cell surface. One PG, syndecan, binds through its intracellular domain to the internal cytoskeleton of the cell, while interacting with another protein - fibronectin - in the extracelluar matirx. Fibronectin also binds other molecules which can regulate cellular growth and other interactions. PGs act like glue in connecting the extracellular and intracellular functions of the cell. Most proteins bind PGs through a PG binding motif of BBXB or BBBXXB where B is a basic amino acid. Some proteins bind to specific sequences in specific GAGs. For instance, antithrombin 3, an inhibitor of blood clotting, binds specifically to heparin, which enhances its interaction with the clotting protein thrombin.

4. Cell Membranes of Eukaryotes

We have studied lipids, proteins, and carbohydrates. Although phospholipid can spontaneously form biliayers, the actual structure of biological membranes is made much more complicated through addition of protein and carbohydrate substituents to the membrane.

We have talked previously about how normally soluble proteins can be made to insert into bilayers by addition of nonpolar attachments. Several examples of such attachments include:

• N-myristoylation (attached myristic acid - 14:0 - through an amide link)
• S-palmitoylation (attach palmitic acid - 16:0 - through a thioester link with a Cys
• farnesyl or geranylgeranyl additon to a CAAX carboxy-terminal sequence in a target protein, where C is Cys, A is aliphatic, and X is any amno acid
• addition of a protein to a glycosyl phophatidylinositol (GPI), through a complex which usually contains a conserved tetrasaccharide core of 3 Man and 1 GlcNAc residues linked to a protein. The GPI can be further modified with extra Gal's and Man, as well as additions to the PI group, which secures the protein in the membrane. GPIs are found in eukaryotic cells, and link many surface antigens, adhesion molecules, and hydrolases to the membrane. GPIs from Plasmoidium falciparum, the malarial parasite which kills about two million people each year, appears to act as a toxin and is the most common CHO modification of the parasite protein.  Mice immunized against  the GPI sequence, NH₂-CH₂-CH₂-PO₄-Man (a1-2) 6Man (a1-2) Man (a1-6) Man (a1-4) GlcNH₂ (a1-6) myo-inositol-1,2-cyclic-phosphate, were substantially protected from malarial symptoms and death after they were exposed to the actual parasite.

Great CHO Web Sites

• Proteoglycans and Glycosoaminoglycans
• Sweet:   a program for constructing 3D models of saccharides from their sequences using standard nomenclature.
• Textbook mistakes in Biochemistry:  Glycogen