See also Drug Deliver where cationic polymers have been sued. 

Naturally derived Cationic Polymers

Cellulose: (C6H10O5)n  is a polysaccharide consisting of a linear chain of several hundred to many thousands of beta(1-4) linked D-glucose units. Cellulose is a fundamental component of the cell wall of plants, constituting the fundamental raw material in such important processes as papermaking. US2006/0242739). 

Chitson: is a natural cationic copolymer composed of randomly distributed N-acetyl glucosamine and D-glycosamine, varying in composition, sequence and molecular chain lenght. Chitosan is one of the most extensively studies cationic polymers as an antimicrobial agent. It possesses inherent antimicrobial activity against many Gram-positive and Gram negative bacterial and fungi at pH<6. Alhtough the exact emchanisms is not fully understood, several mechanisms have been suggested such as changes which occur in the bacterail membrane permeability, breakdon of the cytoplasmic membrane barrier or the blockage of nutrient transport, resulitng in cell lysis. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Cyclodextrins: are sugar derivative pdouced by bacteria grown on starch. 

Dextran: is an FDA approved highly water soluble branch polysaccharide composed of glucose units mianly linked by alpha-1,6-linkages. 

Gelatin: is a natural polymer derived form collagen, commonly applied for pharmaceutical and medical purpsoes because of its biodegradbility and biocompatibility in physiological environments. 

Starch (amylum): is a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. Starch is the principal storage form of carbohydrates in plans. It is accumulated in large quantities in organs such as seeds (wheat, barley, maize, pea, etc) and tubers (potato and yam ) and is a fundamental constituent of the human diet. As a polymer, starch is often used in paper, cosmetic, pharmaceutical and food industries and also used as a basic component for the manufacture of biodegradable plasstics and environment friendly paints (US 2006/0242739).

Synthetically derived Cationic Polymers

The main difficulties with batch to batch variation or natural polymers can be overcome by synthetic polymers. The bioactive moieties and funcitonal groups can be readily incorporated into the synthetic polyemric system to result in specific MW and block structures with degradable linkages if required. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012).

Polyethyleneimine (PEI):  is the most prominent and extensively used cationic polymer containing primary, secondary and tertiary amino functions. It is synthesized in both linerar and branch forms and exists in different MWs. 

Poly(amidoamine)s (PAAs): PAAs are a unique family of synthetic cationic polymers with many desirable properties including biodegradbility, biocompatibility, water solubility and a lower toxicity ompared to other cationic polyerms. PAAs can be obtained by Michael-type polyaddition of primary amines or bis-secondary amines to bis-acrylamides. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Poly(amino-co-ester) (PAEs): are a class of synthetic and hydrolytically degradable polyamines originally developed and investigated as cationic polymers for DNA delivery. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Poly(2-N,N-dimethylaminoethylmethacrylate (PDMAEMA): is one of the most important pH responsive polymers studies to date. It is water soluble cationic polymer. 

Stimuli-Responsive Cationic Polymers

Advanced therapeutic research demands controlled intelligent systems for various therapeutic applicaitons including controlled delivery, nucleic acid separation and other factors. Cationic polymeric systems with various chemical and structural responsive moieties exhibit the property of responsiveness to external stimuli such as temerpature, pH, ionic concentraiton, light, magnetic field, electric field and chemicals. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

pH responsive cationic polymers: The presence of ionizable functional groups on a cationic polymer drametically alters its structural properties at, above and below a specific pH called its pKa. This rapid change in the net charge of pendant or backbone groups with respect to pH casues an alteration of the hydrodynamic volume or conformation of the polymer chains. The pH responsive nature of cationic polymers can be used for biomolecule delivery in neutral or alkaline environments. At a pH above the pKa, the pendant amino groups remain non-ionized leaving the polyemric ahin in a collapsed state, while entrapping the biomolecule. As the pH decreases below the amine pKa, the amine groups become protonated and the polyemric cahin expands due to electrostatis repulsion, in this way releasing the entrapped biomolecules into the surroudning medium. Cationic polymers like chitsoan, PEI, PDMAEMA, PAA have basic functional groups such as primary, seconary and tertiary amine groups that beocme ionized as the pH decreases. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Temperature (thermal) responsive cationic polymers: change their structural proeprties in response to empterature. Tehy can be acheived by incorproating  or grafting temperature resonsive moeities such as pluronic F-127 or poly(N-isopropyl-acrylamide) (PNIPAM) (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Multiresponsive cationic polymers: offer responsiveness to two or more external sitmuli. Dual sensitivity was reproted for a novel triblock copolymer consisting of PAA and PEG which was coverted into an injectable pH and termpature sensitive hydrogel. (Samal, “Cationic polymers and their therapeutic potential, Chem Soc. Rev. (2012)

Ligand gated ion channels/receptors are large multisubunit complexes which are usually pentamers (5 subunits) that form an ion channel in the membrane. The complex may be heteromeric in that it is composed of several different types of subunits or homomeric in that it is composed of a single type of subunit. Each subunit of the complex contains 4 hydrophobic transmembrane domains. The amino and carboxyl termini are each on the cytosolic side of the membrane.

It typically takes 2 molecules of ligand bound to open an ion channel. The ligand binding site is about 1/3 down the way in the channel. The response to this binding is rapid (millisec) enabling ions to flow down their electrochemical gradients. Ligand gated ion channels typically contain allosteric binding sites.

See also Hormones, generally  See also Signal transduction

Hormones may be categorized as lipophilic (fat-soluble), which are nonpolar, or hydrophilic (water-soluble), which are polar. The lipophilic hormones include the steroid hormones and the thyroid hormones.

Hydrophilic hormones are freely soluble in blood, but cannot pass through the plasma membrane of target cells. Their receptors are transmembrane proteins that bind to the hormone outside the cell, and transmit the signal to the interio. In contrast, lipophilic hormones travel in the blood bound to soluble transport proteins. But when they reach a target cell, they can cross the plasma mebrane and bind to intracellular receptors.

Protein Hormones: 

Prolactin (PRL): is released by the anterior pituitary and sitmulates the mammary glands to produce milk in mammals. It also has diverse effects on many other targets, icnluding reulgaiton of ion and water transport across epitheila and activation of parental behaviors.

Glycoprotein hormones: 

Glycoprotein hormones are a group of evolutionary conserved hormones involved in the regulation of reproduction and metabolism. Structurally, the glycoprotein hormones are related heterodimers comprised of a common alpha subunit and a hormone specific beta subunit. This family of hormones includes the following members:

Follicle-stimulating hormone (FSH): is produced by the anterior pituitary and is required for the devleopment of ovarian folicles in feamles. In males, it is required for the development of sperm. FSH stimultes the conversion of testosterone into estrogen in females, and into dihydroxytestosterone in males. FSH adn LH below are collectively referred to as gonadotropins.

Growth Hormone (somatotropin): is released by the anterior pituitary and stimulates the growth of muscle, bond (indirectly) and other tissues, and is also essential for proper metabolic regulation.

Luteinizing hormone (LH): stimulates the production of estrogen and progesterone by the ovaries and is needed for ovulation in femal reproductive cycles. In males, it stimules the testes to produce testosterone, which is needed for sperm production and for the development of male secondary sexual characteristics. It is produced by the anterior pituitary.

Thyroid stimulating hormone (TSH or thyrotropin): TSH is a 28-30 kDa heterodimeric glycoprotein produced in the thyrtrophs of the anterior pituitary gland. TSH stimualtes the thyroid gland to produce the hormone thyroxin, which in turn regulates development and metabolism by acting on nuclear receptors. This hormone controls thyroid function by interacting with the G protein coupled TSH receptor which leads to the stimulation of pathways involving secondary messenger molecules, such as cyclic adenosine 3’5′-monophosphate (cAMP), and ultimately results in the modulation of thyroidal gene expression. Physiological roles of TSH include stimulation of differentiated thyroid functions, such as iodine uptake and the release of thyroid hormone from the gland, and promotion of thyroid growth.

Chorionic gonadotrophin (CG)

Amino Acid Derived Hormones:

Amino acid derivatives are hormones manufactured by enzymatic modificaiton of specific amino acids. They include hormones secreted by the adrenal medulla (the inner portion of the adrenal gland), thyroid and pineal glands. Those secreted by the adrenal medulla are derived form tyrosine. Known as catecholamines, they include epinephrine (adrenaline) or norepinephrine (noradrenaline). Other hormones derived from Trrosine are the thyroid hormones, created by the thyroid gland. The pineal gland secretes a different amine hormone, melatonin, derived form tryptophan.

Norepinephrine (noradrenaline): is an amino acid derived hormone (a catecholamine). Noreipnephrine is secreted into the blood by the adrenal glands, but it is also released as a neurotransmitter by sympathetic nerve endings. Norepinephrine acts as a hormone to coordinate the activity of the heart, liver, and blood vessels druing reponses to stress.

Epinephrine (adrenaline) (see below) and norepinephrine (noradrenaline) are both hormones and neurotransmitters involved in the “fight-or-flight” response, but they differ in their primary actions and effects on the body, with epinephrine primarily affecting the heart and lungs, while norepinephrine mainly focuses on blood vessels.

Peptide Hormones:

Adrenocorticotropic hormone (ACTH or corticotropin) stimulates the adrenal cortex to produce corticosteroid hormones, including cortisol (in humans) and corticosterone (in many other vertibrates). These hormones regulate glucose homeostasis and are important in teh response to stress.

Glucagon: is a hormone that mobilizes glucose as part of the body’s mechanism to control blood glucose. This involves breaking down stored glycogen into glucose and turning on the genes that encode the enymes necessary to synthesize glucose.

Insulin: lowers blood glucose level, stimulates glycogen, fat, and protein synthesis.

Insulin is a hormone that helps regulate the body’s metabolism and blood sugar levels. It is produced by the pancrease and released into the blood when blood sugar levels rise, such as after eating. Insulin assists glucose to enter the cells, where it is used for energy. While glucagon keeps blood glucose from dropping too low, insulin is produced to keep blood glucose from rising too high.

Insulin is a peptide hormone secreted in the body by beta cells of islets of Langerhans of the pancreas and regulates blood glucose levels. Medical treatment with insulin is indicated when there is inadequate production of increased insulin demands in the body. In Type 1 or juvenile diabetes mellitus, pancreatic beta cells are destroyed by the body’s immune system or trauma or injury to the pancreas, leading to decreased or absent insulin production. Patients with type 1 diabetes always require insulin. Type 2 diabetes mellitus is the most common type of diabetes and usually occurs after 45 years. In type 2 diabetes, cells are resistant to the action of insulin. As a result, glucose starts building up in the blood. Treatment with insulin is necessary for the later stages of type 2 diabetes.

Insulin acts by directly binding to its receptors on the plasma membranes of the cells. These receptors are present on all the cells, but their density depends on the type of cells, with the maximum density on hepatic cells and adipocytes.

The role of insulin is thus to lower blood glucose, acting by binding to an RTK. The insulin receptor is a heterotetrameric glycoprotein consisting of two subunits, the alpha and beta subunits. The extracellular alpha subnunits have insulin binding sites. The beta subunits, which are transmembranous, have tyrosine kinase activity. When insulin binds to the alpha subunits, it activates the tyrosine kinase activity in the beta subunit, which causes the translocation of glucose transporters from the cytoplasm to the cell’s surface. Another protein called the insulin response protein binds to the phosphorylated receptor and is itself phosphrylated. The insulin response protein passes the signal on by binding to additional proteins that lead to the activation of the enzyme glycogen synthase, which converts glucose to glycogen, thereby lowering blood glucose.  Other proteins activated by the insulin receptor act to inhibit the synthesis of enzymes involved in making glucose, and to increase the number o glucose transporter proteins in the plasma membrane.

Epinephrine (aka, adrenaline): has diverse effects on different cell types. The distinct effects of epinephrine on different cell types depend on each cell having the same receptor for the hormone, but responding with different signal transduction pathways. In the liver, cells are stimulated to mobilized glucose, while in the heart muscle cells contract more forcefully to increase blood flow. (the fright response is due to release of epienphrine, also called adrenaline, into the bloodstream. Epinephrine receptors in the liver produce cAMP thorugh the enzyme adenylyl cyclase. The cAMP produced activates protein kinases that promote the produciton of glucose from glycogen. In smooth muscle, epinephrine receptors can activate a different G protein that activates the IP3 generating enzyme phospholipase C. As a result, epinephrine sitmulation of smooth muscle results in IP3-regulated release of intracellular calcium, causing muscle contraction.

The sympathetic nervous system (SNS) plays a vital role in maintaining the homeostasis of the body by secreting different neurotransmitters like catecholamines, acetylcholine, glutamine, etc. Among all the neurotransmitters, categholamine has a very important and diversified role in the various organs. Catecholamines contain a categhol (3,4 dihydroxypheynl) group along with an amine group. John Jacob Abel, in 1897 first obtained a crystalline substance form the adrenal gland of sheep in an impure form that can regulated blood pressure, and he named it epinephrine. In 1900, Jokichi Takamine obtained a pure cyrstalline form of epinephrine. He patented it with the name adrenalin. British Approved Name (BAN) introduced the name “adrenaline” in the UK. US Approved Name (USAN) used the term “epinephrine” for this neurotransmitter in the USA. Adrenaline is synthesized form its precursor amino acid tyrosine; it is also synthesized form hepatic hydroxylation of another amino acid, phenylalanine. Synthesis of catecholamine beings with the rate limiting step governed by the enzyme tyrosine hydroxylase, which converts tyrosine into L-DOPA. Subsequently, L-DOPA is coverted to dopamine and then to norepinephrine by DOPA decarboxylase and dopoamine beta-hydroxylase, respectively. Dopamine beta-hydroxylase is a copper containg enzyem and requires ascorbic acid for its function. Both epinephrine and norepinephrine stimulate common receptors name adrenergic receptors. Structurally these receptors have seven hydrophobic transmembrane regions and an intracellular C-terminal domain and an extracellular N-terminalal domain, along with 3 intracellular and extracellular loops. All adrenergic receptros work through different G prtoeins like Gs, Gi,e tc. Beta2 adrenergic receptor is the most abundant and most widely studied adrengergic receptor. (lal, Role of adrenergic receptor signalling in neuroimmune communication” Current Research in Immunology, 2 (2021) 202-217).

Erythropoietin (EPO) is a hormone that belongs to the class of peptide hormones, which are molecules composed of amino acids that act as signaling molecules. It is a naturally occurring hormone which stimulates the production of red blood cells in the bone marrow through a process called erythropoiesis. The production of EPO is useful in treating blood disorders characterized by low hematocrit, which is a low ratio fo red blood cells to total blood cells. Erythropoeisis (EPO): is a process stimulated by the peptide hormone erythropoietin (EPO). Erythropoiesis is the process of red blood cell production, and EPO, produced by the kidneys, stimulates this process. 

Melanocyte-stimulating hormone (MSH) stimulates the snthesis and dispersion of melanin pigment, which darkens the epidermis of some fish, amphibians and reptibles and can control hair pigment color in mammals. It is a peptide hormone of the anterio pituitary.

Parathyroid hormone (PTH): raises blood calcium level by stimulating bone breakdown, stimualtes calcium reabsorption in kidneys and activates vitamin D. Its target tissue is the bone, kidneys and digestive tract.

Steroid Hormones:

Steroid hormones form a large class of compounds, including cortisol, estrogen, progresterone and testosterone, that share a common nonpolar structure. The nonpolar structure allows these hormones to cross the membrane and bind to intracellular receptors.

Steroids are lipids manufactured by enzymatic modifications of cholesterol. Steroid hormones can be subdivided into sex steroids, secreted by the testes, ovaries, placenta, and adrenal cortex, and corticosteroids (mineralocorticoids and cortisol), secreted only by the adrenal cortex.

Estrogens: 

The hormone estrogen has different effects in uterine tissue than in mammary tissue. This differential response is meiated by coactivators and not by the presence or absence of a receptor in the two tissues. In mammary tissue, a critical coactivtor is lacking and the hormone-receptor complex instead interacts with another protein that acts to reduce gene expression. In uterine tissue, the coactivator is present, and can bind the hormone-receptor complex to turn on the expression of genes that encode prtoeins involved in prearing the uterus for pregnancy.

Glucocorticoids are steroid hormones produced from the cortex of adrenal glands. Glcocorticoids are important modualtors of brain function and hypothalamus-pituitary-adrenal (HPA)-axis activity. To reach their central target areas they have to enter the brain by passing the blood-brain barrier (BBB), a dynamic barrier that protects the brain from periopheral influences.

–Cortisol: is a steroid hormone in the glucocorticoid class of hormones and a stress hormone. As medication, cortisol is called hydrocortisone.

Cortisol is a hormone that is produced by the adrenal glands, located on top of the kidneys. The pituitary gland, located in the brain, regulates the amount of cortisol released by the adrenal glands. Cortisol can increase levels of glucose in cells. A number of different genes involved in the synthesis of glucose have binding sties for the hormone receptor complex.

Cortisol prevents the release of substances in the body that cause inflammation. It is thus known to inhibit IL-12, IFN-gamma, IFN-alpha dn TNF-alpha by APCs and TH1 cells. It upregulates IL-4, IL-10 and IL-13 by TH2 cells. This resutls in a shift toward a Th2 immune response.

Testosterone: stimulates development of secondary sex characteristics in males and growh spurt at puberty.

DNA topoisomerases are able to modify the termtiary structure of circular DNA. Prokaryotic enzymes do not require ATP, as do eukaryotic topoisomerases.

DNA topoisomerases have been classified into two types:

Type I DNA topoisomerases: catalyse several reactions, usually in the absence of any high energy cofactor. They remove negative and sometimes positive supercoils from circular DNAs, linkg the single-stranded rings of complementary DNAs, transform single-stranded circles into knotted ones. 

The functions of topoisomerases I are not so well established, alhtough they have been implication in the regulation of transcription and in the transpoisition process (Riou, Eur. J. Biochem, 134, 479-484 (1983). 

Type II DNA topoisomerases: catalyse the reations of relaxation, actenation/decatentation and knotting/uknotting. They are thought to play an important role in repliaction, transcription, recombination and repair processes. 

Polymerases are enzymes which polymerize new DNA or RNA using an existing DNA or RNA template. Polymerases are essential in DNA replication and transcription.

PARPS  See outline

Tankyrasses (TANKs)

TANKs were identified as components of the human telemeric complex. They have also been proposed to have a role in vesicale trafficking and may serve as scaffolds for proteins involved in various other cellular processes. TANks are (ADP-ribose) transferases with some features of both signalling and cytoskeletal proteins. They contain the PARP domain, which catalyses poly-ADP-ribosylation of substrate proteins, the sterile alpha motif, which is shared with certain signalling molecules and the ANK domain, which contains 24 ankyrin repeats homologues to the cytoskeletal protein anykyrin. The ANK domain interacts with a telomeric protein, Telomere Repeat binding Factor-1 (TRF-1). These proteins were thus named TRF1-interacting ankyrin-related ADP-ribose polymerase (TANKs).

Functions: One of the more specific functions of TANK is the ADP-ribosylation of TRF-1. Human telomer function requires two telomere specific DNA binding proteins, TRF-1 and TRF-2. TRF-2 protecs chomosome ends and FR-1 regualtes telomere lenght. ADP ribosylation inhibits the ability of TRF-1 to bind to telomeric DNA. This poly-ADP-ribosylation of TRF-1 releases TRF-1 from the telomeres, opening up the telomeric complex and allow acces to telomerase. Thus, TANK functions as a positive regulator of telomere lenght, allowing elongation of the telomeres by telomerase.  

Members: Tankyrases (TANKs) members include TANK-1, TANK-2 and TANK-3.

Telomerase

Telomerase is a ribonucleoprotein responsible for the template independent synthesis of telomeric DNA. Human telomerase is made up of two components: (1) an essential structural RNA (TER) (wehre the human component is referred to as hTER), and (2) a catalytic protein (telomerase reverse transcriptase or TERT) (wehre the human component is referred to in the art as hTERT).

Telomerase works by recognizing the 3′ end of DNA, e.g., telomeres, and adding multiple telomeric repeats to its 3′ end with the catalytic protein component (e.g., hTERT) which has polymerase activity, and hTER which serves as the template for nucleotide incorporation. Telomerase recognizes the tip of a G rich strand of an existing telomere DNA repeat sequence on the parental strand and elongates it. The telomerase is able to do an RNA template that is a component of the enzyme itself (much like the reverse transcriptases of viruses). After extension of the parental strand by telomerase, replication of the lagging strand can be completed. The replication is such though that the parental strand still protrudes out since all of the extended parental strand is not replicated. This protrusion loops back to tuck its singlestranded terminus into the duplex DNA of the telomeric repeat sequence creating a unique structure which protects it from degradative enzymes.

Telomerase is also important in aging.

An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule called “the reductant” or “electron donor” to another molecule called “the oxidant” or the “electron acceptor”. This group of enzymes typically uses NADP or NAD+ as cofactors.

Peroxidases (EC number 1.11.1.x): when combined with H2O2 and a halide (chloride, bromide, iodide) form a potent cytotoxic system which contributes to the host defense against invading microorganisms and possibly tumor cells. Neutrophils and monocytes contain the same peroxiase (myeloperoxidase, MPO) and eosinophils a different peroxidase (eosinophil peroxidase, EPO), in cytoplasmic granules and these enzymes are dischared into the pahgosome following particle ingestion.

1. Myeloperoxidase (MPO, donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7): is a tetrameric, heavily glycosylated basic (PI 10) heme protein of about 150kDa. It is compirsed of two identical disulfide linked protomers, each of which possesses a protoporphyrin containing 59-64 kDa heavy subunit and a 14 kDa light subunit. MPO is a complex heme protein which possesses multiple intermediate states, each of which are influenced by the availability of reduced oxygen species such as O2- and H2O2 and nitric oxide (NO, nitrogen monoxide). At ground state, MPO exists in the ferri (Fe(III) form. Upon addition of H2O2, the heme group of MPO is oxidized two e- equivalents forming a reactive ferryl pi cation radical intermediate termed Compound I.

MPO amplifies the oxidizing potential of H2O2 by using it as a cosubstrate to generate a variety of reactive oxidants and diffusible radical species. Formation of phenoxyl radicals by MPO, such as during oxidation of the free amino acid L-tyrosine, can promote protein cross-linking and oxidation of LDL lips. MPO may also use H2O2 and nitrite (NO2-), a major end product of NO metabolism, to generate a microbicidal oxidant capable of nitrating phenolic compounds and proteins in vitro. NO2- is ubiquitous in biological tissues and flues. During inflammatory and infectious processes where NO production is enhance, palsma and extracellular lelvels of NO2- are markedly increased. Schmitt (Biochemistry, 1999, 38, 16904-16915).

–Where MPO is found: MPO is abundant in azurophil granules in neutrophils and is promptly dischared after activation by different agonists. The heme protein is sorted in primary azurophilic granules of leukocytes and secreted into both the extracellular milieu and the phogolysosomal compartment following phagocyte activation by a variety of agonists.

Intracellular MPO is found in granulocytes in amounts which are generally constant from cell-to-cell, and the rate adn intesity of color change can accordingly be employed to accurately reflect the level of granulocytes in a blood sample being assayed. However, becasue MPO appears as an intercellular component of a granulocyte, those granulocytes present in the sample being assayed must be lysed or disrupted to release the enzyme before the sample is treated with the peroxide and the dye to make a meaningful determination of granulocyte level (US6,046,019).

–MPO activity may be determined by any of a variety of standard methods such as a colorimetric based assay where a chromophore that serves as a substrate for the peroxidase generates a product with a characteristic wavelenght which may be followed by any various spectroscopic methods including UV visible or fluorescence detection. 

Based on MPO catalyzed oxidation of different dyes, several spectrophotometric methods to assay MPO have been reported. These methods provide satisfactory measurements of MPO activity and can be used for quantitation of the number of PMNs in pure preparations. However, problems can be encountered when applying these methods to determine MPO activity in PMN containing tissues (Xia, Analytical Biochemistry 245, 93-96 (1997)). 

Xia, Analytical Biochemistry 245, 93-96 (1997) report a method for quantitation of MPO activity in PMN containing tissues which uses a one step purification on a Sephadex G075 gell filtration chromatography to separate myoglobin from tissue MPObased on the large difference between the MW of MPO, 118 kDa and myoglobin, 16 kDa,  followed by a spectrophotometric assay of MPO. 

–MPO Mass: is readily deteremined by an immunological method (e.g., ELISA). MPO mass in a sample can also be determined indirectly by in situ peroxidase staining of the bodily sample. 

Neuraminidases (also known as sialidases): are enzymes that have been identified in many viruses, bacteria and eukaryotes that cleave sialic acid moieties and can be involved in many functions in vivo. It has been shown that neuramindases can play a significant role in the pathogenesis of infectious diseases, whose etiologic agents produce neuraminidase to cleave sialic acids in infected tissues to facilitate their ability to invade a host. (US2006/0241063). Neuraminidases cleave terminal sialic acid residues from cell surface molecules such as glycoproteins and glycolipids. As a result of this cleavage, internal sugar residues can be exposed that are normally protected and not available to pathogens. Neuraminidase activity can be particularly important for bacterial adhesion to mucosal surfaces. Mucous typically is highly sialyated and can be a major component of innate mucosal immunity. In mucosal diseases, commensal bacteria are separated from epithelial cells by a mucous barrier. Pathogenic bacteria have been shown to produce sialidases which can decrease the viscoity of the mucous and thus enable the bacteria to colonize on the epithelial cell membrane.

Neuraminidase Inhibitors

Neu5Ac2en is typically known as a sialidase or neuraminidase inhibitor. A preferred group of inhibitors are similar in structure to Neu5Ac2en. For example, Neu5Ac2en is known as the lead compound for the synthesis of one of the most well known sialidase inhibitors, zanamivir (Relenza) (US 2006/0241063).

An example of a neuradminidase inhibitor that has been aprroved for the treatment of human influenza isoseltamivir (Tami-flu). It is a syntehtic sialic acid analog that has been modified at the C4 position.

See also ABO blood antigens   See also removing A and B antigens from Type A, B, and AB cells

Glycosidase refers to an enzyme that cleaves a covalent bond between sequential sugars in a glycan or between the sugar and the backbone moiety (e.g., between sugar and peptide backbone of glycoprotein).

Alpha-glucosidase (GAA): breaks down glycogen, a larger molecule, into glucose. 

A person with Pompe’s disease has significantly reduced levels of GAA, or no GAA at all and so is unable to break down glycogen into glucose. That inability results in glycogen accumulating in the muscles of affected patients in excessive amounts. Pompe’s diease is found in two forms -early oset and late onset. Early onset presents shortly after birth. Glycogen accumulates in the pateint’s heart and skeletal uscles, cuasing a progressive deterioration of the heart muscles. Without treatment, a patient with early onset Pompe’s diesase will die form cardiac or respiratory failure before reaching one year of age. 

Following the dsivoery that Pompe’s disease is assocaited with GAA deficiency, researchers tried injecting the enzyme. However, it was taken up by the patient’s liver, reducing glycogen levels there but not in the skeletal or heart uscles where the excess glycogen does the most harm. Later researchers overcame this problem by modifying the injected GAA to include mannose-6-phosphate (M-6P) which promotes GAA uptake in heart and skeletal muscle cells containing M-6-P receptors, including the cells that failed to take up GAA in prior treatment attempts. By 1997, the FDA approved Duke University’s application for Orphan Drug Designation for a new therapy for Pompe’s disease based on the injection of a recombinant form of GAA. See US Patent Nos. 7,351,410 and 7,655,226. 

Plant-derived enzymes

Alpha-galactosidase (E.C. 3.2.1.22); alpha-D-galactoside galactohydrolase): catalyzes the hydrolysis of the terminal linked alpha-galactose moeity from galatose-containing oligosaccarides. These include, for example, the naturally occurring disaccharide melibiose (6-O-alpha-D-galactopyranosyl-D-glucose), the trisaccharide raffinose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-f- ructofuranoside) and the tetrasaccharide stachyose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-galactopyranosyl-(1-6)-O-alpha0 a-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside).

Alpha -galactosidases have potential use in various applications. They may hydrolyze alpha-galactose residues form polymeric galactomannans, such as in guar gum which has been used to imporve the gelling properties of the polysaccharide. Alpha-galactosidase can also hydrolyze raffinose from beet sugar syrup, which can be used to facilitate the sugar crystallization from molasses, since the raffinose presents an obstacle to the normal cyrstallization of beet sugar. Alpha-galactosidase can also be used to hydrolyze stachyose and raffinose in soybean milk, thereby reducing or eliminating the undesirable digestive sid eeffects which are associated with soybean milek. The enzyme can also remove the temrinal alpha-galactose residue sfrom other glycans, such as the erythrocyte surface antigen conferring blood group B specificity. This has potential medical use in transfusion therapy by converting blood group type B to unersal donr type O.

Endoglycosidases:

EndoS: The Fc portion of IgG contains a conserved glycan on each heavy chain attached to Asn-297. This oligosaccharide is of the complex biantennary type with a core fucose linked to the innermost N-acetylglucosamine (GlcNAc). These glycans are located in the interface between the CH2 domains (second constant domain of the heavy chains). EndoS is an endoglycosidase secreted by the human pathogenStreptococcus pyogenes. EndoS specifically hydrolyzes the asparagine-linked glycan on IgG between the two core GlcNAc residues. In contrast to many related endoglycosidases that require or are enhanced by denaturation of the glycoprotein substrate, EndoS only hydrolyzes native IgG.

Bjorck (US 2010/0135981) discloses that EndoS is useful in treating diseases such as arthritis and idiopathic thrombocytopenic purpura (ITP) mediated by IgG antibodies by the administration of EndoS polypeptide which hydrolyzes IgG in blood (deglycosylation of IgG). The deglycosylation of IgG by EndoS abrogates its arthritis inducing capacity in mice.

Exoglycosidases

Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids.

Alpha-N-acetylgalactosaminidases (EC 3.2.1.49) are used for destroying A antigens of blood cells.

–N-acetyl-alpha-D-galactosaminidase: from the domestic chicken, Gallus domesticus, is an important exoglycosidase which degrades the human blood group A epitope. The isolated enzyme has a MW of 49.1 kDa and is highly selective for PNP-N-acetyl-alpha-D-galactosaminide. The enzyme hydrozyes the teminal N-acetyl-alpha-D-galactosaminide reisudes form blood group A2 erythroctyes. Protease activity is below detectable limits. The enzyme has a pH optima of 3.7, a pI of 8.15 and is relatively unaffected by ionic strenght and is stable at 4C (Hata, Biochem Int 1992, 28(1): 77-86).

Alpha-galactosidases (EC 3.2.1.22): are used for destroying B antigens of blood cells.

–Alpha-D-Galactosidase: from coffea canephora is an important exoglycosidase which degrades the human blood group B epitope. The isolated enzyme has a MW of 36.7. The isozyme is highly selective for alpha-D-galactosides. It hydrozyles the terminal alpha-D-galactosyl residue from the blood group B epitope. (Haibach, Biochem Biophys Res Commun 1991, 181(3): 1564-71)

The glycosyltransferases are a family of some 250-300 different intracellular, mebrane-bound enzymes that participate in the coordinate biosynthesis of the glycostructures of polypeptides, including glycoproteins, proteoglycans and glycolipids. They are classified into groups based on their nucleotide monosaccharide donor specificity. For example, the galactosyltransferases use UDP-galactose as the activated monosaccharide donor whereas the sialytransferases use CMP-sialic acid and the fucosyltransferases use GDP-fucose. (Hueller, US14348822)

Examples of Glycosyltransferases

Examples of glycosyltransferases include galacosyltransferases, fucosyltransferases, glucosyltransferase, N-acetylgalactosiminyltransferases, N-acetylglucosaminyltransferases, glucuronyltransferases, sialyltransferanses, mannosyltransferases, and oligosaccharyltransferases. 

Prokaryotic glycosyltransferases include enzymes involved in synthesis of lipooligosaccharides (LOS), which are produced by many gram negative bacteria. The LOS typically have terminal glycan sequences that mimic glycoconjugates found on the surface of human epithelial cells or in host secretions. 

Large scale enzymatic synthesis of oligosaccharides depends on the availability of sufficient quantities of the required glycosyltransfereases. Glycosyltransferases can be used as regio- and steroselective catalysts for the in vitro synthesis of oligosaccharides. However, production of glycosyltransferases in sufficient quantities for use in preparing oligosaccharide structures has been problematic. WO98/54331 discloses modified nucleic acids which encode glycosyltransferases having enhanzed hield . 

Serine proteases are a large family of proteoytic enzymes that include digestive enzymes, trypsin and chymotrpsin, components of the complement cascade and of the blood clotting cascade, and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. They are so named because of the presence of serine residue in the active catalytic site for protein cleavage. They have a wide range of substate specificites and diverse biological functions. However, a common catalytic mechanism is shared among several sub-families through a very similar tertiary structure supported by a highly conserved catalytic triad of serine, histidine and asparate. The active site structure of one serine protease, subtilisn, is among the most studied and best understood (US 2006/0088883).

Serine proteases such as elastase and cathepsin G degrade collagen, proteoglycans and elastin, a protein found in walls of arteries. Additionally, these proteases contribute to activation of matrix metalloproteinases, which cause further destruction of tissue. Serine proteases are associated with numerous other chronic inflammatory disease states. In inflamed joints, serine proteases lead to the destruction of collagen and proteoglycans of articular cartilage associated wtih arthritis. Serine proteases also degrade fibrinogen and fibronectin, proteins involved with homeostasis. Loss of homeostasis can lead to uncontrolled coagulation, fibrinolysis, and inflammation, resulting in sepsis. Serine proteases also aid the spread of HIV because these proteases cleave HIV proteins into active peptides, necessary for proper replication. Serine proteases have a highly reactive serine in their active site. Many proteases are secreted as inactive precursors that can be activated locally when needed. For example, plasminogen is an inactive protease precursor in the blood that is cleaved locally by other proteases called plasminogen activators to yield the active serine proteaseplasmin which helps break up blood clotes. Tissue type plasminogen activator (tPA) is often given to patients who have had thrombotic stroke to help dissolve the arterial.

Examples of Serine Proteases

Plasmin: is a potent serine protease that degrades firbrin into fibrin degradation products. Plasmin is formed from the proteolytic cleavage of the zymogen plasminogen. Plasmin formation is catalyzed by the actions of the two major mammalian activators: tissue-Plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Since t-PA is able to activate plasminogen to plasmin, it is capable of initiating the cascade of events needed to dissolve undesired blood clots. Urokinase type plasminogen activator is a multi domain protein with one domain being a trypin like serine rpotease. This protease domain converts plasminogen to plasmin by cleavage at an arginyl residue. The fibrin clot provides a surface to increase the efficiency of plasmin generation through formation of a ternary complex of fibrin, t-PA and plasminogen. As a result, fibrinolysis almost exclusively occurs on the clot surface and not in the circulation.

PAs are controlled by specific inhibitors such as PA inhibitor type-1 (PAI-1) which inactivates both urokinase-type PA (uPA) and tissue-type PA (tPA). Reduced levels of PAI-1 may result in creased fibrinolysis and an associated bleeding diathesis.

Elastase: also known as human leukocyte elastase (HLE) and human neutrophil elastase (HNE) is a serine protease released from the azurophilic granules of the neutrophile as part of the normal inflammatory response. NE is capable of degrading key structural elements of connective tissue, such as elastin, collagen, and proteoglycan. Under normal hoemeostatic conditions, the protease inhibitor alpha1-antitrypsin serves as an important regulator of proteolysis by HLE, thereby preventing damage of the lung alveolar matrix.

–Neutrophil elastase (NE): is a major proteinase in primary granules in neutrophils that participates in microbicidal activity. NE (Leukocyte elastase, EC 3.4.21.37), a serine endopeptidase, is characterized by serine in the active tripeptide catalytic site: Asp, His, Ser. NE is the most abundant of foru serine proteases present in neutrophils, which also include proteinase 3, cathepsin G,a nd neutrophil serine protease. 

NE is a 29.5 kD protein stored in mature form in the azurophilic granules of neutrophils and is present at high concentraitons per azurophilic granule. 

Although NE proteinase activity is critical for normal innate immune funciton, rellease of NE into the airway mileiu contributes to lung disease progression. (Shinbashi, “Neutrophil elastase and chronic lung disease” Biomolecules, 2021). 

Complement protein B: is a single polypeptide chain serum glycoprotein that carries the catalytic center of the alternative pathway C3-convertase. It is a novel type of serine protease characterized by an unusual structure in the NH2-terminal region of its catalytic fragment Bb, when compared to other serine proteases. Assembly of the biomolecular,C3bBb, C3-convertase proceeds in two well defined steps, First, B binds stoichiometrically to C3b in a reaction requiring Mg2+ or Ni2+ ions. Second, complement protein D catalyzes the cleavage of a single arginyllysyl peptide bond of B resulting in the release of fragment Ba and the formation of the C3bBb protease. For a discussion of the alternative pathway click here.