2-Doschak PH310-Aby Structure and B-cell Diversity (2023) ver2.pdf

Pharmacy 310 Antibody Structure and B-cell Diversity Dr. Michael R. Doschak Professor Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Email: [email protected]  Antibodies (Aby) Y-shaped glycoproteins,...

Pharmacy 310 Antibody Structure and B-cell Diversity Dr. Michael R. Doschak Professor Faculty of Pharmacy & Pharmaceutical Sciences University of Alberta Email: [email protected]  Antibodies (Aby) Y-shaped glycoproteins, assembled from 4 polypeptide chains – 2 Identical “heavy” H chains (~50 kDa) – 2 Identical “light” L chains (~25 kDa) Chains are linked by disulfide bonds Each polypeptide chain composed of: – Variable region (N-terminus) – Constant regions (remainder) Figure 2-2  Antibodies Individual Aby are specific Human Aby repertoire 1016 Aby is the secreted form of Immunoglobulin (Ig) Each B-cell expresses just one specificity of Ig on it’s surface When bound by Antigen (Ag), B-cell differentiates to Aby-secreting plasma cell Figure 2-1  Aby Functional Regions Ascertained by protease digestion Plant protease “papain” cleaves Aby into 3 fragments: – 2 Fab (Fragment antigen binding) – 1 Fc (Fragment crystallizable) Gut protease “pepsin” degrades Fc fragment, and leaves F(ab’)2 fragment Fc on intact Aby important for opsonization Figure 2-3 part 1 of 2 Figure 2-3 part 2 of 2  Antibody Isotypes An isotype refers to related proteins and/or genes from a particular gene family The "immunoglobulin isotype" (or class of Aby) refers to the genetic variations (or differences) in the constant regions of the heavy and light chains  Antibody Isotypes Defined by different H-chain C regions Five isotypes: IgG, IgA, IgM, IgE, IgD (GAMED) Heavy chain denoted by corresponding lower-case Greek letter: γ, α, μ, ε, δ Light chains have only 2 isotypes: Kappa (κ) and Lambda (λ) Figure 2-4  Variable and Constant Domains Ig subunits 100-110 amino acid in length Termed Immunoglobulin domains V-region - single variable domain – VH : in the Heavy chain – VL : in the Light chain C-regions – little or no amino acid sequence diversity Antibody Tertiary Structure Figure 2-5 Antibody Tertiary Structure Figure 2-6  Antigen-binding Site Also known as “paratope” Framework regions (FR) Hypervariable region (HV) loops, or Complementarity Determining Regions (CDR) CDRs provide a binding surface complementary to the antigen Figure 2-7 part 1 of 2 Figure 2-7 part 2 of 2  Antigenic Determinants Antibody binds to Antigen on parts termed Antigenic Determinants, or “epitopes” Usually carbohydrates, protein, or both (eg, glycoprotein, polysaccharide, glycolipid, peptidoglycan) Most Antigens are multivalent (many potential epitopes) Figure 2-9  Antibody Binds Antigen due to Molecular “Complementarity” And Non-Covalent binding Complementarity permits tight, highly- specific macromolecular binding (protein-protein; protein-nucleic acid; Aby drug-receptor) “Lock and Key” model There is a Complementarity of: shape, charge, polarity, hydrophobicity  Organic Molecules of Cells (i.e., Nucleic Acids, Proteins, Sugars, Lipids Information carried by organic macromolecule is expressed by means of weak noncovalent bonds:  Organic Molecules of Cells (i.e., Nucleic Acids, Proteins, Sugars, Lipids Information carried by organic macromolecule is expressed by means of weak noncovalent bonds: Ionic bonds Hydrogen bonds Hydrophobic effect Van der Waals attractions Epitopes Bind to Pockets, Grooves, Surfaces Figure 2-10  DNA codes for Protein Antibodies are Proteins Segments of DNA (termed Genes) code for proteins Coded by base-pair sequences A = Adenine T = Thymine C = Cytosine G = Guanine  DNA codes for Protein Antibodies are Proteins Segments of DNA (termed Genes) code for proteins Eukaryotic cells have both “coding” and “non-coding” segments in genomic DNA blueprint Coding: Exons Non-coding (or intervening): Introns  RNA Transcription Antibody Primary RNA transcript contains introns (blue) Antibody Messenger mRNA transcript Codes for polypeptide  mRNA Translation to Polypeptide Chain Carboxy Amino Terminus Terminus Amino Carboxy Terminus Terminus  Protein – Tertiary Structure Antigenic Ligand (Dark Blue) Antigen Binding Loop created by Helix to Sheet β-sheet Transition (in blue) α-helix (in red) Polypeptides greater than 40 or 50 amino acids are termed proteins  Post-translational Events Polypeptide Glycosylation – Attachment of sugars (in Endoplasmic Reticulum & Golgi) N-glycosylation – at Asparagine residues (Asn) in sequence Asn-X-Thr O-glycosylation – at Serine (Ser) or Threonine (Thr) Polypeptide Sorting and Targeting by cell – Proteins for Secretion, versus: – Proteins for insertion into phospholipid bilayer of intracellular compartments, for Cell Membrane expression  Immunoglobulin Diversity Generated “before” encounter with Ag Ig genes organized different to other genes Ig genes “fragmented” in all cells – cannot be expressed (except in the B-cell) Consist of families of gene segments (Ig gene “locus” or “loci”) Termed the germline configuration  Figure Ig Germline 2-14 Genes Segments: V-variable; J-joining; C-constant; D-diversity (and L-leader peptides)  Gene Segment Recombination Recombination enzymes RAG-1 and RAG-2: – Recombination Activating Gene (RAG) enzymes (found only in lymphocytes; “both” B- and T-cells) – Recombination process also uses other enzymes present in all nucleated cells (used to repair DNA breaks)  Gene Segment Recombination Ig Constant region genes are “ready to transcribe” (but they still consist of introns & exons) In contrast, Ig Variable region genes are coded for by 2 or 3 gene loci that require RAG-mediated enzymatic rearrangement of the germline DNA Figure 2-15 part 1 of 2  Gene Segment Recombination Recall that Ig Variable domains have three HV regions The 3rd HV region is in fact coded for by a spliced “junction” between – V and J for Light Chain – V, D, and J for Heavy chain – Thereby increasing Variable domain diversity There are also anchoring (or Membrane Coding, MC) exons Figure 2-15 part 2 of 2 Class Demonstration of Immunoglobulin Gene Splicing and Transcription  Germline Ig Genes Figure Generate 2-14 Aby Diversity Segments: V-variable; J-joining; C-constant; D-diversity (and L-leader peptides) Figure 2-16 200 120 10,530 Recombination Activating Gene (RAG) Enzyme mediated Gene Segment Recombination Process directed by DNA base pair sequences called Recombination Signal Sequences (RSS) Two RSS types: – Heptamer (CACAGTG) – Nonamer (ACAAAAACC) Separated by 12 and 23 base pair “spacers” (12/23 rule) Recombination Signal Sequences Figure 2-17 RAG Complex uses the 12/23 Rule Figure 2-18 part 1 of 3 RAG cuts DNA at heptamer sequences Figure 2-18 part 2 of 3 Figure 2-18 part 3 of 3  Naïve B-cells express IgM and IgD V, D, J rearrangements trigger transcription of full Ig gene mRNA is differentially spliced, then translated to polypeptide chain μ and δ heavy-chain C region genes the first to be transcribed and expressed Accomplished by differential mRNA splicing Each B-cell produces Ig of only one specificity by “Allelic exclusion” (no further V rearrangements)  Figure CONSTANT REGION 2-20 ISOTYPE GENE CLUSTER IgM and IgD Transcribed first, and together LONG 1o RNA TRANSCRIPT (BOTH IgM and IgD SEGMENTS), DIFFERENTIAL SPLICING AND EXON REMOVAL Figure 2-21 part 1 of 2 Figure 2-21 part 2 of 2  Ig Assembly and surface expression Separate Ig polypeptides enter endoplasmic reticulum Self-assemble into Ig molecules Hydrophobic MC (Membrane Coding) region inserts into bilipid membrane Ig also associates with disulfide-linked transmembrane proteins, Igα and Igβ (serve to signal the B-cell that surface Ig has bound Ag) Surface Ig also termed the B-cell receptor (BCR) Figure 2-23 Surface and Secreted Ig Forms derived by same Figure 2-24process  Aby Affinity Maturation Somatic hypermutation (in rapidly dividing B-cells) further increases diversity of rearranged gene segments Results in phenomenon of “affinity maturation” (ie, over time, Aby becomes more specific for the triggering antigen)  Aby Isotype Switching IgM and IgD are co-expressed on B-cells through differential mRNA splicing of the primary RNA transcript IgM is secreted first, as a pentamer Isotype switching occurs after further DNA recombination events that rearrange the same V-region to be used with other “downstream” heavy-chain Constant genes Most common switch is to IgG, which is secreted as a monomer IgE is also secreted as a monomer IgA is secreted as a dimer (using the J, or joining chain) V-region specificity the same on all isotypes Different C-regions give different effector functions  Isotype Switching: FurtherFigure 2-27 events recombination M D G3 G1 A1 G2 G4 E A2 A1 A1 Figure 2-26 Figure 2-30 Figure 2-28 Attributes of Different  Figure 2-29 part 1 of 2 Antibody Effector  Figure Antibody Isotype 2-29 part 2 of 2 Figure 2-32  Resource Materials Parham P; The Immune System, 2nd Edn., Garland Science, New York, 2005. Lodish H, et al; Molecular Cell Biology, 5th Edn., W.H. Freeman and company, New York, 2004. (www.whfreeman.com/lodish) Alberts et al.; Molecular Biology of the Cell, Garland Publishing, New York, 1983. Freifelder D; Essentials of Molecular Biology,Jones and Bartlett, Boston, 1985. © Copyrighted material contained herein is reproduced under ss. 29-29.4 of the Canadian Copyright Act. This document is available for your individual use; further distribution may infringe copyright.