Complex Carbohydrates

Suggested readings: B-S pp. 337-346, 11.3, 11.4
Overview of diverse types of complex carbohydrates and their cellular and extracellular functions.
Complex processing of oligosaccharide chains on proteins will be discussed later with protein synthesis and sorting.

Oligosaccharides

Oligosaccharides are long carbohydrate chains.
They can be homopolymers or heteropolymers.
Glycogen, starch, and cellulose are homopolymers of glucose.
Heteropolymers contain two or more monosaccharides and/or modified monosaccharides in their chains, examples include hyaluronic acid and heparin.
Glycoconjugates are complex carbohydrates that consist of oligosaccharide chains linked to proteins or lipids.
- Two classes of molecules containing both protein and oligosaccharide chains:
  - Proteoglycans (aka mucopolysaccharides): mostly carbohydrate, composed of multiple carbohydrate chains anchored to a small protein core.
  - Glycoproteins: mostly protein, have one or more attached oligosaccharide chains.
- Molecules containing one or more sugar moieties attached to a lipid are called glycolipids.

Location and Function of Glycoconjugates

Most glycoconjugates are extracellular or associated with the cell membrane.
Many plasma proteins are glycoproteins.
Proteoglycans function as lubricants and structural components in connective tissue, and mediate adhesion of cells to the proteins of the extracellular matrix.
Glycoproteins and glycolipids are found as constituents of the plasma membrane; the carbohydrate portion faces the extracellular space.
Glycoconjugates are involved in binding reactions, including those of pathogenic viruses and bacteria.
Carbohydrate structure provides specificity for blood group antigens and for binding by cellular adhesion molecules or lectins.

Free Oligosaccharides

Free oligosaccharides (without either protein or lipid attached) are found in many biological fluids including milk and seminal fluid.
Oligosaccharides in milk may protect the infant from pathogenic bacteria.
Oligosaccharides in seminal fluid may promote fertilization.
Fucose is a six-carbon deoxysugar (lacks one –OH group).

Glycosaminoglycans

Glycosaminoglycans are anionic (negatively charged) polysaccharide chains composed of repeating disaccharide units.
One of the modified sugars in the disaccharide repeat is usually glucosamine or galactosamine.
Negative charges come from attached sulfur groups.
Hyaluronic acid is unsulfated; the negative charges come from the carboxyl groups of the sugar acids.
Heparin is a highly sulfated polymer with both N- and O-sulfate groups, which acts as an anticoagulant.

Proteoglycans

Proteoglycans contain multiple glycosaminoglycans attached to a core protein forming a "bottle brush" type structure.
The older term for proteoglycans is mucopolysaccharides.
Proteoglycans are found in all connective tissues and extracellular matrices and are attached to the surface of many cells.
Because of their high content of charged polysaccharides, proteoglycans are highly hydrated.
The swelled, hydrated structure of proteoglycans is largely responsible for the volume of the extracellular matrix and permits diffusion of small molecules between cells and tissues.
Hyaluronic acid is unsulfated and not incorporated into proteoglycans.
Hyaluronic acid provides much of the "jelly" of the umbilical cord and serves to protect the umbilical blood vessels from damage during fetal movement.

Hyaluronic Acid and Fertilization

Hyaluronic acid is also a major component of the extracellular matrix of the cells that surround the oocyte.
During the acrosome reaction, activated sperm secrete hyaluronidase, which hydrolyses the hyaluronic acid and permits the sperm to penetrate to the oocyte.
When preparing oocytes for intracytoplasmic sperm injection (ICSI), embryologists use commercially prepared hyaluronidase to facilitate removal of the cumulus and corona radiata cells from the oocytes.

Glycoproteins

The carbohydrate chains of glycoproteins can be either O-linked (to the hydroxyl group of serine or threonine) or N-linked (to the side chain amino group of asparagine).
Many proteins found in serum are glycoproteins.
Elastase, a glycoprotein serine protease secreted by neutrophils, has two branched oligosaccharide chains.

Synthesis of O-linked Glycoproteins

Synthesis of O-linked glycoproteins occurs by adding sugar residues one at a time after the protein is synthesized.
The process involves glycosyl transferases, which transfer the glycosyl moiety from an activated UDP-sugar to the protein-bound oligosaccharide.
UTP, which is used to synthesize the UDP-sugar, is a nucleotide similar to ATP.

Mucins

Mucins are a type of O-linked glycoproteins that are produced by epithelial cells.
Some are secreted (as in the mucus of nasal secretions); others remain membrane-bound and contribute to cell-cell interactions.
Mucins are large glycoproteins with a high content of serine, threonine, and proline residues and numerous O-linked oligosaccharide chains.
The hydrated carbohydrates are responsible for the viscous nature of these molecules, which provide a protective physical layer at the surface of the epithelium.

N-Linked Glycoproteins

The second class of glycoproteins has one or more oligosaccharide chains N-linked to an asparagine residue.
All N-linked oligosaccharides have a common pentasaccharide (5 sugar) core.
The core structure contains three mannose molecules attached to two molecules of N-acetyl-glucosamine.

N-Linked Glycoprotein Structure

Diagrammed structures of oligosaccharide chains often use abbreviations for the monosaccharide constituents.
The core of the N-linked oligosaccharide is composed of Man (Mannose) and GlcNAc (N-acetylglucosamine).
Diagrams also specify the linkage between sugar moieties ( $\beta$ -1,4, $alpha$ -1,6, etc.)

Synthesis of N-Linked Glycoproteins

The oligosaccharide chains of N-linked glycoproteins are synthesized on a specialized lipid carrier called dolichol phosphate.
The process involves several different mechanisms for activating the individual sugars, and enzymes on both sides of the endoplasmic reticulum.
A fourteen-sugar oligosaccharide is transferred to the protein chain as the latter is being synthesized.
The oligosaccharide chain is then modified by removal of some of its sugar residues and (in some cases) addition of others.

Oligosaccharides as Antigens

Oligosaccharides are antigens, meaning that particular oligosaccharide structures are recognized by and bind to antibody proteins triggering immune response.
Recognition of certain oligosaccharide structures as “foreign” to the body provides a mechanism for host protection against pathogenic microorganisms.
Interpretation of “self” as “foreign” can cause major problems.

ABO Blood Groups

One example of variation in oligosaccharide structures within the human population is the blood groups A, B, and O.
All three have a common oligosaccharide core, which is found attached to both glycoproteins and glycolipids on the surface of erythrocytes and other cells.
The O antigen has the basic four-sugar structure.
The A antigen is formed by the addition of GalNAc to this structure.
The B antigen is formed by the addition of an unmodified galactose instead of the N-acetylgalactosamine.

Genetics of Blood Type

Expression of the ABO blood types is determined by the genes for the terminal glycosyltransferase which adds the last GalNAc or Gal to the basic structure.
The A allele codes for an enzyme which adds GalNAc; the B allele codes for the enzyme which adds Gal.
The O allele gene product does not produce an active glycosyltransferase.
Each individual has two alleles for blood type (one gene from each parent) and can thus be AA, AO, BB, BO, AB, or OO.
Individuals who are AO and AA produce antigen A.
Individuals who are BB or BO produce antigen B.
AB individuals produce both; OO individuals produce neither.
Individuals who do not produce either the A or B antigen will recognize that antigen as foreign.
If an individual with type A blood is given a transfusion of type B blood, the red cells will clump together or agglutinate because antibodies are formed to the B antigen.
Each antibody can simultaneously bind to the surface of two red blood cells, causing clumping.
Since all individuals can synthesize the O antigen, antibodies are not formed to it.
Individuals with the OO genotype are “universal donors”.
Individuals with the blood type AB are universal recipients.

Lectins

Lectins are a class of plasma membrane-bound proteins that recognize specific oligosaccharides on the surface glycolipids and glycoproteins of other cells.
They mediate a variety of transient cell-cell adhesion processes, including those occurring in sperm-egg interactions, blood clotting, lymphocyte recirculation, and inflammatory responses.
The lectins include two classes of cell-adhesion molecules (CAMs) called selectins and cadherins.
L –selectin has a role in the attachment of the embryo to the endometrium of the mother’s uterus trigger a signaling cascade for implantation.

Glycosidases

Glycosidases are enzymes that remove individual monosaccharides from oligosaccharide chains (involved in digestion or breakdown of the chains).
Cellular glycosidases are localized within specific organelles called lysosomes.

Lysosomal Storage Diseases

There is a class of genetic storages diseases in which there is inadequate enzymatic activity of one or another lysosomal glycosidase.
The result is the accumulation, within the lysozyme, of the undigested substrate.
One example of a lysosomal storage disease is Tay-Sachs, which is caused by a deficiency in a particular glycosidase called hexosaminidase A.
The resultant accumulation of glycolipid is accompanied by major neurological damage and is fatal in early childhood.

Pre-implantation Genetic Screening

Pre-implantation genetic screening was used successfully at EVMS to assure a healthy pregnancy to a couple who had previously lost a child to Tay-Sachs (one in four chance of subsequent unscreened pregnancy).