✓ 2 Differentiate among the types of glycoproteins in regard to structure and function.

Let us look at a glycoprotein present in the blood serum that has dramatically improved treatment for anemia, particularly that induced by cancer chemotherapy. The glycoprotein hormone erythropoietin (EPO) is secreted by the kidneys and stimulates the production of red blood cells by bone marrow, the tissue in interior of bones. EPO is composed of 165 amino acids and is N-glycosylated at three asparagine residues and O-glycosylated on a serine residue (Figure 10.17), making the mature EPO 40% carbohydrate by weight. Glycosylation enhances the stability of the protein in the blood; unglycosylated protein has only about 10% of the bioactivity of the glycosylated form because the protein is rapidly removed from the blood by the kidney. The availability of recombinant human EPO has greatly aided the treatment of anemias. Some endurance athletes have used recombinant human EPO to increase the red-blood-cell count and hence their oxygen-carrying capacity, a practice prohibited by most professional sports organizations. Drug-testing laboratories are able to distinguish some forms of prohibited recombinant EPO from natural EPO in athletes by detecting differences in the glycosylation patterns through the use of isoelectric focusing.

The proteoglycan aggrecan and the protein collagen are key components of cartilage. The triple helix of collagen provides structure and tensile strength, whereas aggrecan serves as a shock absorber (Figure 10.20). The protein component of aggrecan is a large molecule composed of 2397 amino acids (Figure 10.21). Many aggrecans are linked together by hyaluronate, a glycosaminoglycan. The aggrecan molecule itself is decorated with the glycosaminoglycans chondroitin sulfate and keratan sulfate. This combination of glycosaminoglycan and protein is especially suited to function as a shock absorber. Water is absorbed on the glycosaminoglycans, attracted by the many negative charges. Aggrecan can cushion compressive forces because the absorbed water enables the proteoglycan to spring back after having been deformed. When pressure is exerted, such as when the foot hits the ground in walking, water is squeezed from the glycosaminoglycan, cushioning the impact. When the pressure is released, the water rebinds. Osteoarthritis, the most common type of arthritis, can result from the proteolytic degradation of aggrecan and collagen in the cartilage in the joints due to inflammation. More typically, the cause is simple “wear and tear.” Glucosamine and chondroitin (Figure 10.18) are widely promoted as a treatment for osteoarthritis, but the benefits to anyone but the producers and marketers of the supplement are very ambiguous.

Although glycosaminoglycans may not seem to be familiar molecules, they are common throughout the biosphere. Chitin is a glycosaminoglycan found in the exoskeleton of insects, crustaceans, and arachnids and is, next to cellulose, the second most abundant polysaccharide in nature (Figure 10.22). Cephalopods, such as squid, have razor sharp beaks, which are made of extensively cross-linked chitin, to disable and consume prey.

The third class of glycoproteins that we examine comprises the mucins (mucoproteins). In mucins, the protein component is extensively glycosylated to serine or threonine residues by N-acetylgalactosamine (Figure 10.7). Mucins are capable of forming large polymeric structures and are common in mucous secretions. These glycoproteins are synthesized by specialized cells in the tracheobronchial, gastrointestinal, and urogenital tracts. Mucins are abundant in saliva where they function as lubricants.

A model of a mucin is shown in Figure 10.23A. The defining feature of the mucins is a region of the protein backbone termed the variable number of tandem repeats (VNTR) region, which is rich in serine and threonine residues that are O-glycosylated. Indeed, the carbohydrate moiety can account for as much as 80% of the molecule by weight. A number of core carbohydrate structures are conjugated to the protein component of mucin. Figure 10.23B shows one such structure.

Mucins adhere to epithelial cells and act as a protective barrier; they also hydrate the underlying cells. In addition to protecting cells from environmental insults, such as stomach acid, inhaled chemicals in the lungs, and bacterial infections, mucins have roles in fertilization, the immune response, and cell adhesion. Mucins are overexpressed in bronchitis and cystic fibrosis, and the overexpression of mucins is also characteristic of adenocarcinomas—cancers of the glandular cells of epithelial origin.

The human ABO blood groups illustrate the effects of glycosyltransferases. Each blood group is designated by the presence of one of the three different carbohydrates, termed A, B, or O, on the surfaces of red blood cells (Figure 10.24). These structures have in common an oligosaccharide foundation called the O (or sometimes H) antigen. The A and B antigens differ from the O antigen by the addition of one extra monosaccharide, either N-acetylgalactosamine (for A) or galactose (for B) through an α-1,3 linkage to a galactose moiety of the O antigen.

Specific glycosyltransferases add the extra monosaccharide to the O antigen. There are three common genes encoding such glycosyltransferases, and each person inherits the genes for each type from each parent. The type A transferase specifically adds N-acetylgalactosamine, whereas the type B transferase adds galactose. These enzymes are identical in all but 4 of 354 positions. The O phenotype is the result of a mutation in the O transferase that results in the synthesis of inactive enzyme.

These structures have important implications for blood transfusions and other transplantation procedures. If an antigen not normally present in a person is introduced, the person’s immune system recognizes it as foreign. Adverse reactions can ensue, initiated by the intravascular destruction of the incompatible red blood cells.

Why are different blood types present in the human population? Suppose that a pathogenic organism such as a parasite expresses on its cell surface a carbohydrate antigen similar to one of the blood-group antigens. This antigen may not be readily detected as foreign in a person with the blood type that matches the parasite antigen, and the parasite will flourish. However, other people with different blood types will be protected. Hence, there will be selective pressure on human beings to vary blood type to prevent parasitic mimicry and a corresponding selective pressure on parasites to enhance mimicry. The constant “arms race” between pathogenic microorganisms and human beings drives the evolution of diversity of surface antigens within the human population.

Although the role of carbohydrate attachment to proteins is not known in detail in most cases, data indicate that this glycosylation is important for the processing and stability of these proteins, as it is for EPO. Certain types of muscular dystrophy can be traced to improper glycosylation of dystroglycan, a membrane protein that links the extracellular matrix with the cytoskeleton (Section 1.4). Indeed, an entire family of severe inherited human diseases called congenital disorders of glycosylation has been identified. These pathological conditions reveal the importance of proper modification of proteins by carbohydrates and their derivatives.

An especially clear example of the role of glycosylation is provided by I-cell disease (also called mucolipidosis II), a lysosomal storage disease. Lysosomes are organelles that degrade and recycle damaged cellular components or material brought into the cell by endocytosis. Normally, a carbohydrate marker directs certain digestive enzymes from the Golgi complex to lysosomes where they function. In patients with I-cell disease, lysosomes contain large inclusions of undigested glycosaminoglycans and glycolipids, the “I” in the name of the disease. These inclusions are present because the enzymes normally responsible for the degradation of glycosaminoglycans are missing from affected lysosomes. Remarkably, the enzymes are present at very high levels in the blood and urine. Thus, active enzymes are synthesized, but, in the absence of appropriate glycosylation, they are exported instead of being sequestered in lysosomes. In other words, in I-cell disease, a whole series of enzymes is incorrectly addressed and delivered to the wrong location. Normally, these enzymes contain a mannose 6-phosphate residue as a component of an N-oligosaccharide that serves as the marker directing the enzymes from the Golgi complex to lysosomes. In I-cell disease, however, the attached mannose lacks a phosphate. I-cell patients are deficient in the N-acetylgalactosamine phosphotransferase catalyzing the first step in the addition of the phosphoryl group; the consequence is the mistargeting of eight essential enzymes (Figure 10.25). I-cell disease causes the patient to suffer severe psychomotor retardation and skeletal deformities, similar to those in Hurler disease. Remarkably, mutations in the phosphotransferase have also been linked to stuttering. Why some mutations cause stuttering while others cause I-cell disease is a mystery.