Biochemistry 9 Copy 2 (3) PDF

25 Duha Al-Nader Hala Abu-Dyouk Nafiz Abutarboush 1  immunoglobulins IgM Class § Location: Mainly intravascular (blood & lymph), B-cell surface (monomer). *IgM can be present in different forms: monomer (Y shape), pe...

25 Duha Al-Nader Hala Abu-Dyouk Nafiz Abutarboush 1  immunoglobulins IgM Class § Location: Mainly intravascular (blood & lymph), B-cell surface (monomer). *IgM can be present in different forms: monomer (Y shape), pentamer (most common), hexamer (least common) § Known Functions: Primary immune response (1st produced). § Primary role in antigen agglutination (ex. ABO). § IgM only exists as a monomer on the surface of B cells. § Monomeric IgM has a very low affinity for antigen. § A J-chain is involved in the process of multimerization. § Cm4 mediates multimerization (Cm3 may also be involved). *An antibody is a tetramer (consist of 4 polypeptide chains) that might consist of 1 antibody ,2 antibodies, or 6 antibodies that’s how it represents itself in plasma. The process of IgM multimerization: *Structure - function relationship - IgM has 4 constant regions and 1 variable region. - Antibodies are joined together by the formation of disulfide bond between the end of constant region of one antibody and the constant region of another. -The process is facilitated by the J-chain a polypeptide (sequence of amino acids) that brings molecules into close proximity allowing the formation of disulfide bond. Once all five monomers are joined J-chain is retained within the pentameric IgM. 2 *The final molecule in its multimer form (whether a pentamer or a hexamer) involves only one Joining chain. C Cystine IgG Class § Location: Blood, lymph, intestine. *Most abundant immunoglobulin in blood. § Produced in response to a wide variety of antigens, (ex. bacteria, viruses). § Found only as a monomer. § Only immunoglobulin that crosses placenta. § Known Functions: § Responsible for secondary immune response. § The predominant antibody produced in the 20 immune responses. § Provides the major line of defense for the fetus & during first few weeks of newborns. § Coats organisms to enhance phagocytosis by neutrophils and macrophages (opsonization). IgA Class § Structure & location: (found as a multimer) § Plasma →monomer, dimer (most common form), or trimer. § Secondary immune response § Secretions (tears, saliva, intestines, milk, bronchial secretion, urine, sweat) →dimer attached to “secretory component” which provides protection against digestive enzymes. 3  Known Functions: § Localized protection (respiratory, urinary tract and bowel infections). § Provides immunity to infant’s digestive tract & body (translocated). § Protection against pathogens before interning blood circulation. § The process of dimerization -The problem of having antibodies (proteins) in these spaces is that they’re affected by environment (PH, Temperature) of these spaces, their structure must be protected to perform their function, so they’re linked in their most common form Dimer via the formation of S-S bond facilitated by J-chain. IgA & Transcytosis *Underneath epithelial tissue we have lymphatic tissue containing lymphocytes. - B-lymphocytes (under epithelium) synthesize antibodies that enter epithelial cells & then they’re released into spaces to provide protection against foreign bodies that enter these spaces. - The secretory component is produced by epithelial cells lining mucous membranes & provides protection against digestive enzymes in secretions. - B cells located in submucosa produce dimeric IgA, then the secretory component coils around it. - The complex is then internalized by epithelial cells. - The internalized complex is transported across epithelial cells into mucosal environment. - Antibody is associated with secretory component & remains in this form in secretion 4  Expression outside Stalk protein is digested Internalization IgD Class § Location: B-cell surface (primarily), blood, and lymph. § Known Functions: § In serum: function is unknown. § On B cell surface: initiate immune response. IgE Class § Location: - Blood & bound to mast cells and basophils throughout body. -present in the form of a monomer. Known Functions: § Allergic reactions (histamines and heparin): increased vascular permeability, skin rashes, respiratory tract constriction (wheezing), and increased secretions from epithelium (watery eyes, runny nose) § Possibly lysis of worms, parasitic infections trigger the production of IgE & so IgE is involved in destruction of worms. 5 Immunological memory - If an antigen enters your body IgM is produced, it recognizes the antigen through its variable and hypervariable regions that contain specific sequence of amino acids that determine the antibody’s ability to bind to antigen’s epitope. - The idea of secondary immune response, if you get exposed to the same antigen ever again you are immune and response will be stronger and much faster compared to primary response. - IgM & IgG binding is highly specific and might recognize the same antigen, but they differ in constant region of heavy chain which determines the class of antibody. 1) Primary immune response - IgM is the first antibody produced during initial exposure to an antigen, followed by IgG production. - IgM levels reach the peak within the first 1-2 weeks and decline quickly. 6 2) Secondary immune response - Involves the production of memory cells. - It elicits the production of IgM at a lower magnitude compared to IgG - IgG production is stronger since it is responsible for secondary immune response. -In second exposure to the same antigen IgG is produced rapidly and in higher quantities compared to first exposure. IgG concentration remains elevated for an extended period as it has long half-life (compared to IgM). IgG concentration decreases as the antigen is no longer present. Class (Isotype) switching -Changing the production of immunoglobulin from one type to another retaining the same antigen specificity. -During this process, the constant region of the antibody’s heavy chain is changed, while the variable region of the heavy chain stays the same(unchanged), for example, switching from the IgM-specific μ region to the IgG-specific γ region. - isotype switching in memory cells, the constant region can be switched to different isotype, while the variable region, which determines antigen specificity, remains unchanged & accordingly it becomes capable of producing different isotypes that target the same antigen. § Antibodies with identical specificity but of different classes § Generated in a chronologic order in response to the antigen § Gene rearrangement: movement of VDJ from a site near one C gene to a site near another C gene 7 - In the variable region there are 3 genes in heavy chain (variable, diversity, joining) and 2 genes in light chain, these genes undergo rearrangement or recombination to generate an amino acid sequence complementary to the target antigen. *There are 5 types of heavy chain constant region in antibodies & according to these types they’re classified into: IgA, IgM, IgG, IgE, IgD. Diseases that affect gamma globulins Diseases that affect antibodies either lead to high overproduction (indicating cancer), or underproduction § Myelomas (cancer affecting plasma cells): increased production § Multiple myeloma: a neoplastic condition, increase in one class, or a particular light chain (Bence Jones protein) § Decreased production may be restricted to a single class or may involve underproduction of all classes (ex. agammaglobulinemia) § These diseases ae related to immunity Electrophoresis -sample of plasma is placed in semisolid gel. + -is based on the motion of charged particles under the effect of an electric field toward an electrode of opposite charge. -molecules have differing mobilities based on their charge, shape, and size. - Lane A represent normal pattern of serum protein electrophoresis in a healthy individual, while Lane B shows a different pattern as the γ-band is wide & prominent (indicating pathological condition). -Albumin band is the darkest and most prominent and - is the fastest moving protein in serum protein electrophoresis due to its small size & weight and high abundance/concentration. 8 -Common proteins such as α (α1, α2) and β (β1, β2) aren’t single proteins they’re collection of proteins that have similar MW and appear to be in the same band. *α, β, γ are globulins. *Remember: gamma represents immunoglobulins(antibodies). Densitometer representation - Represents density and quantity of a molecule - Albumin has a high sharp peak since it’s present in high concentrations and is the most abundant plasma protein, whereas gamma has a wide peak as it involves different types of cells(immunoglobulins) of different MW. § The graph compares the normal pattern (Green) with multiple myeloma (Red). - Abnormal spike in gamma region indicates an overproduction of a specific type of cell (cancer affecting one type of cell), while others remain the same. - If there is an overproduction of all gamma globulin proteins then the peak will be round not sharp. 9  Plasma proteins § Plasma is the liquid part of blood where cells are suspended, which means they’re insoluble & don’t form hydrogen bonds with the liquid they’re contained in. § Cellular part of blood is called packed cell volume / hematocrit (Adult male: 47 %, Adult females: 42%) - Blood contains everything since it’s the interface between external environment and body tissues. - To obtain cells from blood we use centrifugation, which separates different components of a blood sample based on their density. - When a blood sample is left for some time, cells precipitate because there is no constant movement of cells. § Top layer – supernatant soluble plasma (proteins, dissolved gases, electrolytes, wastes, nutrients, small molecules such as glucose & urea). § Middle layer – Buffy coat consisting of WBCs & platelets. § Bottom layer (highest density) – Hematocrit (RBCs). 10 ESR (erythrocyte sedimentation rate) - An increase in ESR indicates a pathological condition. - An indicator of pregnancy, ESR increases due to physiological changes during pregnancy. -Percentage of packed cell volume in a 100ml of blood § In males – 47% § In females – 43% water blood solids (Most of them are plasma proteins). Plasma § Liquid medium where cells are suspended § Composition: ▪ Water (92%) ▪ Solids (8%) Organic components: § Plasma proteins: Albumin, Globulins & Fibrinogen § Non-protein nitrogenous compounds: urea, free amino acids, uric acid, creatinine, creatine & NH3 § Lipids: Cholesterol, TG, phospholipids, free fatty acids § Carbohydrates: Glucose, fructose, pentose § Other substances as: Ketone bodies, bile pigments, vitamins, enzymes &hormones Inorganic components: Na+, K+, Ca2+, Mg2+, Cl-, HCO3-, HPO42-, SO42- 11 - Hemoglobin is a corpuscular protein found inside red blood cells(erythrocytes). - Hemoglobin, albumin and fibrinogen (has an elongated structure that aids its function) are all proteins but they differ significantly in structure. Plasma proteins are a mixture: § More than 500 plasma proteins have been identified. § Normal range 6-8 g/dl (the major of the solids) § Some of these proteins are simple and some are conjugated (bound to something else either a carbohydrate -glycoprotein- or a lipid -lipoprotein-). ü The graph displays the logarithmic concentration of various proteins in blood plasma. ü Classical plasma proteins have higher concentrations compared to interleukins and cytokines which are present in much lower concentrations. - All plasma proteins are glycosylated (bound to carbohydrate), except albumin which constitutes 50-60% (high concentration) of plasma proteins, if glycosylated, plasma becomes very thick (addition of carbohydrate increases viscosity). Separation of plasma proteins ▪Salting-out (ammonium sulfate): -It depends on the concept of increasing salt concentration to fractionate proteins. 12 -As the salt is added it ionizes into +ve & -ve charges that interfere with protein binding to water (proteins are soluble due to hydrogen bonding), since salts have higher preference to water compared to proteins. -Depending on protein solubility, proteins of lower solubility exit first & proteins of higher solubility exit last, forming 3 different groups: fibrinogen, albumin, and globulins. ▪Electrophoresis (most common): serum (defebrinated plasma-clotting factors and fibrinogen are removed), five bands (albumin, α1, α2, β, and γ). *Fibrinogen is removed in electrophoresis due to its large size that clogs the gel. Densitometer representation Albumin has a tall sharp peak since it constitutes 50-60 % of plasma proteins & a concentration of 35-55 g/l. ‫ﺗﻣت ﻛﺗﺎﺑﺔ ھذا اﻟﺷﯾت ﻋن روح واﻟدة زﻣﯾﻠﻧﺎ ﻋﻣرو راﺋد ﻣن دﻓﻌﺔ ﺗﯾﺟﺎن‬ ‫دﻋواﺗﻛم ﻟﮭﺎ ﺑﺎﻟرﺣﻣﺔ واﻟﻣﻐﻔرة‬ Thank you 13