SISTEMA CLIMÁTICO GLOBAL

1.6.2.1 Structure, function and physiology.

Human serum transferrin belongs to the transferrin superfamily which also contains o VO transferrin which is found in egg white and lactoferrin, which is found in milk and white blood cells. The transferrins specifically bind free Fe^"^ ions at physiological pH in serum and transport Fe^"^ ions into cells via transferrin-receptor mediated uptake (Baker and Lindley, 1992).

Human serum transferrin is a 79 kDa glycoprotein which is synthesised primarily in the liver and contains two N-glycosylation sites (Figure 1.24). The mature protein has a polypeptide chain of 679 amino acids but a pre-protein containing a 19 amino acid hydrophobic signal sequence is found in the liver. The transferrin molecule contains 19 disulphide bonds and has two distinct lobes which are termed the N- and C-lobes, inter-connected by a small sequence of amino acids. Each lobe has been further subdivided into two domains termed N1 and N2, and C l and C2 respectively (Baker and Lindley, 1992). Since, both lobes have very similar sequence homology

Chapter 1. Introduction

and conformations, it is probable that transferrin originated by genetic sequence repetition (Williams, 1982). Similarly to ai-antitrypsin, transferrin is also an extremely polymorphic protein with an excess of 30 distinct genetic variants having been described (Welch and Longmead, 1990).

N-lobe C-lobe

an io n binding site Iron b inding site

G lyco syla tio n site

413

G lyco syla tio n site

611

Figure 1.24 Ribbon view of the human serum transferrin showing Fe^ and C0 3^ (an ion) and N-linked glycosylation sites.

Human transferrin contains two N-linked glycans at asparagines 413 and 611 (Figure 1.24), although in other mammals the number of glycans can vary from one to four, as in rabbit and bovine transferrins, respectively (Baker and Lindley, 1992). In all species the glycans are located on the C-lobe of the molecule. The glycans found on human serum transferrin are predominantly complex biantennary ones (82±3%), although smaller amounts contain one hi- and one triantennary (17±2%) and two triantennary complex glycans (>1%). Although transferrin is not an acute phase protein, the microheterogeneity of the glycan structures has been shown to increase during liver disease and pregnancy (Montreuil e ta l, 1997).

C h apter 1.___________________________________Introduction

Studies carried out on partially or completely deglycosylated transferrin, or on recombinant transferrin after site-specific mutagenesis of N-glycosylation sites have demonstrated that the glycosylation state of the molecule does not affect its Fe^"^- binding capacity or recognition by the plasma membrane-bound transferrin receptor (Montreuil et a l, 1997). Van Eijk and colleagues (1987) has also shown that microheterogeneity of the glycans, which varies from zero to five sialic acids residues per glycan, also does not affect its Fe^^-delivery capacity to rat reticulocytes.

The total human body concentration of Fe^^ is approximately 3-5 g and is distributed amongst four major compartments; 1.5-3 g in the red blood cells, 0.1-0.3 g bound to proteins such as myoglobin, cytochrome c, sulphur proteins, peroxidases and catalases and approximately 1 - 1.5 g bound to ferritin mainly in the liver. The transferrin-bound Fe^^ in the serum and lymph tissues is only 3-4 mg, which in relation to the total Fe^^ in the human body, points to the efficiency o f transferrin in maintaining the low levels of iron in the circulation (Montreuil et a l, 1997).

Iron-saturated transferrin is termed /lo/o-transferrin and takes on a pink coloration while iron-free transferrin is termed û/?o-transferrin. Transferrin has the ability to bind two Fe^^-ions together with two CO]^ anions, the binding of the CO]^ anions being a prerequisite for the binding of the Fe^^ ions. The binding site for each Fe^^ -ion is found deep within the inter-domain cleft. The amino acids involved in the binding of the Fe^^ ion include two tyrosine, one histidine and one aspartic acid residue; tyrosines 95, 188, histidine 249, aspartic acid 82 in the N lobe and tyrosines 426, 517, histidine 585, aspartic acid 392 in the C-lobes. The CO^' -anion fits into the cleft containing the Fe^^ ion and is held in place by a series of hydrogen bonds between the anion, an arginine and an adjacent threonine residue (arginines 124 and 426, in the N- and C-lobes, respectively (Baker and Lindley, 1992)).

The binding and release o f Fe^^ -ions involve large changes in the conformation of the transferrin molecule, with the apo-transferrin having an open lobed conformation while the Fe^"^ -bound conformation has both lobes closed. At neutral pH, transferrin binds Fe^^ and COs^ -ions very tightly and also binds to the transferrin receptor on the plasma membrane o f cells (Montreuil et a l, 1997). The transferrin molecule delivers its cargo Fe^^ -ions to the cell by internalisation of the transferrin-receptor

C h apter 1.___________________________________Introduction

complex into the cell. The molecule then releases the Fe^^ and COs^' ions owing to a fall in pH to 5.6 in the acidic environment of the endosome (Hirose, 2000). The mechanism by which this process occurs has been termed the dilysine trigger mechanism and is thought to occur by protonation o f lysine residues 206 and 296 in the N-lobe at low pH (Hirose, 2000). Thorstensen and Romslo (1990) have shown that cellular uptake o f the Fe^^ ion from transferrin results in the reduction o f the iron molecule to its Fe^^ state.

1.6.2.2 Transferrin and human disease.

Hereditary atransferrinaemia, a deficiency of plasma transferrin, is an extremely rare condition and has only been reported in 8 patients (Bentier et a l, 2000). Atransferrinaemia is characterised by paleness and extreme fatigue with microcytic anaemia, which results from diminished incorporation o f iron into haemoglobin (7- 55% o f normal) (Goya et a l, 1972). Untreated patients have also been described with low iron in the marrow, hepatomegaly, recurrent infections, marked deposits o f iron in the liver, pancreas, thyroid, myocardium and the kidneys.

Treatment of atransferrinaemia was initially carried out using plasma transfusions but this was eventually replaced by infusions of purified transferrin to reduce the risk of infectious diseases (Schwick, 1977). Treatment with augmentation therapy every two weeks is well tolerated and patients respond well and return to haemotologically normal levels. Iron stores in ferritin and the liver remain high but excess iron can be removed through phlebotomy (Beutler et a l, 2000).

The underglycosylation of transferrin and other serum glycoproteins in CDG patients is described in section 1.5. However, underglycosylation of serum glycoproteins is also evident in patients with long term or chronic alcohol dependence (Henry et a l,

1999). Henry and colleagues have shown that transferrin, a%-antitrypsin, haptoglobin, clusterin and serum-amyloid protein are all underglycosylated in the plasma of alcoholic patients (1999). The underglycosylation o f glycoproteins has been used in the detection of long term alcohol abuse or in the monitoring o f its treatment. As a point of interest the discovery o f CDG-I is based on a serendipitous observation made

C h apter 1.___________________________________Introduction

by Jaeken and colleagues who noted similar plasma transferrin lEF patterns between alcoholic patients and those patients suffering with neurological and multi-system defects (Jaeken et a l, 1984). Transferrin has also been implicated in the reinforcement of the immune system and therefore constitutes an important defence mechanism in the body (Rossen, 1966).

C h apter 1.___________________________________Introduction