Immunoglobulins as Binding Proteins Lecture 10, Medical Biochemistry

Lecture 10, Outline Structures of Immunoglobulins Functional and Structural Differences between class members The immunoglobulin fold motif in immunoblobulins and other binding proteins

Immunoglobulin Function The primary function of immunoglobulin molecules are to bind "foreign" proteins, carbohydrates, nucleic acids and glycolipids so that they can be cleared from the organism by cells specialized to perform such processes. Immunoglobulins typically bind antigens with K d values in the picomolar to micromolar range. As part of this function, immunoglobulins are found in extracellular fluids, a variety of bodily secretions, and attached to the outer surface of cell membranes

Immune System Overview

Each immunoglobulin has two binding sites

Immunoglobulin Structure Immunoglobulins consist of two types of polypeptides, H chains for "heavy" molecular weight and L chains for “light” molecular weight.

Bence-Jones Proteins Structures of immunoglobulins (IG) were first deduced using "Bence-Jones" proteins (IG molecules) isolated from urine and plasma of patients with multiple myeloma. In this disease, a single B lymphocyte proliferates uncontrollably and develops into a large clonal pool of essentially identical cells. Each cell secretes the same IGs into the plasma, allowing isolation of large amounts of one protein. In addition, excessive amounts of L chains are produced and passed into the urine. Sequencing of these L chains from different patients was the first insight into IG structure.

L Chains –L chains are usually about 25,000 kDa in size. In different antibodies or Bence-Jones proteins, each L chain that has been sequenced shows two recognizable regions: – 1. A C-terminal region (about half) that is nearly constant in most immunoglobulins. Slight variations in this region produce 2 isotypes called kappa (  ) and lambda ( ). – 2. An N-terminal region that varies considerably from one immunoglobulin to the next.

H Chains The sizes of H chains varies with immunoglobulin class. IgM - Immunoglobulin M - early plasma antibody response from B lymphocytes - mu (µ) type H chains. Also displayed on membrane surface of these cells by means of a slightly different H chain. IgG - predominant later plasma antibody response from B lymphocytes -  -type H chains. IgA - secretory fluid antibodies -  -type H chains. IgE - anti-parasite antibodies (also involved in allergic reactions) -  -type H chains. IgD - specific function unknown -  -type H chains.

H-chains (cont) H chains also have variable and constant regions in their amino acid sequences. The constant region is the C-terminal three- fourths of the polypeptide. Slight variations of this region also produce H chain isotypes among the different antibody classes. The variable region is the N-terminal one-fourth of the polypeptide.

Note on Immunoglobulin Genetics The genetic mechanism that results in H and L chain structure causes the attachment of a different variable region sequence to each constant region in the immunoglobulin produced by each different type of B lymphocyte. This sequence difference causes the wide variation in binding specificity found in antibody populations. How immunoglobulin diversity is ensured by recombinational and transcriptional mechanisms at the genetic level will be discussed later in the course.

Immunoglobulin Classes

Immunoglobulin M

Variable Region - Antigen Binding Site

Vitamin K Bound in an Antigen Binding Site

Papain Cleavage of IgG: IgG molecules treated for a limited time with the protease papain can be cleaved into 2 kinds of fragments. Under these conditions, papain only hydrolyzes the H chain at a single peptide bond located in the "hinge" region between two immunoglobulin fold domains.

Papain Fragments (cont) Each IgG so treated yields 3 fragments F c comprising the linked C-termini of the 2 H chains. This region does not bind antigen, but can bind proteins found in complement, a complex of proteins from plasma that target cells containing bound antibodies. Two molecules of F ab, comprising the N-terminal half of each H chain linked to its respective L chain by a disulfide bond. Each F ab fragment contains 1 binding site for antigen or hapten. Because the F ab fragment is monovalent (has only one antigen-binding site), it cannot form precipitates with polyvalent antigens, even though it can still bind to the antigen.

Immunoglobulin Structure (cont)

Structure of Immunoglobulin Fold Motif

Space-Filling IgG Structure

Other binding proteins having the immunoglobulin fold motif

Examples: Binding Proteins with the Immunoglobulin Fold Motif –1. T-cell receptors such as CD8 and CD4 (involved in AIDS pathogenesis, for example). –2. The major histocompatibility complex (MHC) proteins, involved in tissue typing and a variety of immune system functions. –3.  2-microglobulin, which interacts with Class I MHC proteins and plays a role in their functions. –4. Neuronal cell-adhesion molecule (N-CAM), a protein that is important in cell-cell and cell- extracellular substratum interactions.