Hyaluronan synthases (HAS) are membrane-bound

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

s that use UDP-α-''N''-acetyl-D-glucosamine and UDP-α-D-glucuronate as substrates to produce the

glycosaminoglycan Glycosaminoglycans (GAGs) or mucopolysaccharides are long, linear polysaccharides consisting of repeating disaccharide units (i.e. two-sugar units). The repeating two-sugar unit consists of a uronic sugar and an amino sugar, except in the case o ...

hyaluronan Hyaluronic acid (; abbreviated HA; conjugate acid, conjugate base hyaluronate), also called hyaluronan, is an anion#Anions and cations, anionic, Sulfation, nonsulfated glycosaminoglycan distributed widely throughout connective tissue, connective ...

at the

cell surface The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extra ...

and extrude it through the membrane into the

extracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...

space.

Isoforms

There are three mammalian hyaluronan synthases described to date -

HAS1 Hyaluronan synthase 1 is an enzyme that in humans is encoded by the ''HAS1'' gene. Structure Hyaluronan or hyaluronic acid (HA) is a high molecular weight unbranched polysaccharide synthesized by a wide variety of organisms from bacteria to mamm ...

, HAS2, and HAS3. Each of these

isoforms A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have uniqu ...

resides at a different chromosome location and has been

cloned Cloning is the process of producing individual organisms with identical genomes, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction; this reproduction of an organism by itself without ...

. Two of the main differences between the isoforms are the chain length of the

molecules that they produce and the ease with which they can be released from the cell surface. When mammalian cells are stimulated by changes in their immediate environment (

cytokines Cytokines () are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B cell, B lymphocytes, T cell, T lymphocytes ...

extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix (ICM), is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and bio ...

proximities), the HAS isoforms respond differently and appear to be under different control mechanisms. During the development of the

embryo An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sp ...

, each isoform is uniquely expressed, both spatially and temporally. * HAS2 is probably the most important synthase at this time as mice lacking the ability to express HAS2 (

knock-out mice A knockout mouse, or knock-out mouse, is a genetically modified mouse (''Mus musculus'') in which researchers have inactivated, or "Gene knockout, knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They ...

) die at mid-

gestation Gestation is the period of development during the carrying of an embryo, and later fetus, inside viviparous animals (the embryo develops within the parent). It is typical for mammals, but also occurs for some non-mammals. Mammals during pregn ...

, * HAS1 or HAS3 knock-out mice show no effect on foetal development. The isoforms of HAS also display varying physiological effects and therapeutic potentials. HAS2 is overexpressed in

breast cancer Breast cancer is a cancer that develops from breast tissue. Signs of breast cancer may include a Breast lump, lump in the breast, a change in breast shape, dimpling of the skin, Milk-rejection sign, milk rejection, fluid coming from the nipp ...

cell lines and is associated with

lymph node A lymph node, or lymph gland, is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that includ ...

metastasis Metastasis is a pathogenic agent's spreading from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, ...

, while HAS1 and HAS3 lack any correlations with cancer development or metastasis. HAS-2 has also been proposed as a nanotherapeutic agent to combat

osteoarthritis Osteoarthritis is a type of degenerative joint disease that results from breakdown of articular cartilage, joint cartilage and underlying bone. A form of arthritis, it is believed to be the fourth leading cause of disability in the world, affect ...

in joints displaying synovial

inflammation Inflammation (from ) is part of the biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The five cardinal signs are heat, pain, redness, swelling, and loss of function (Latin ''calor'', '' ...

as a result of increased hyaluronan depolymerization. Contrastingly, stimulation of HAS3 has been linked to increased inflammation and atheroprogression by means of increased

interleukin Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that are expressed and secreted by white blood cells (leukocytes) as well as some other body cells. The human genome encodes more than 50 interleukins and related ...

release and

macrophage Macrophages (; abbreviated MPhi, φ, MΦ or MP) are a type of white blood cell of the innate immune system that engulf and digest pathogens, such as cancer cells, microbes, cellular debris and foreign substances, which do not have proteins that ...

activation.

Structure

HAS1 has a single

catalytic Catalysis () is the increase in reaction rate, rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst ...

domain A domain is a geographic area controlled by a single person or organization. Domain may also refer to: Law and human geography * Demesne, in English common law and other Medieval European contexts, lands directly managed by their holder rather ...

and is embedded in the

transmembrane A transmembrane protein is a type of integral membrane protein that spans the entirety of the cell membrane. Many transmembrane proteins function as gateways to permit the transport of specific substances across the membrane. They frequently u ...

channel to form

glycosidic A glycosidic bond or glycosidic linkage is a type of ether bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. A glycosidic bond is formed between the hemiacetal or hemiketal group of a ...

linkages in the production of hyaluronan (HA). It contains five transmembrane helices and three interface helices whose overall architecture resembles a tepee. The ''

Pasteurella multocida ''Pasteurella multocida'' is a Gram-negative, nonmotile, penicillin-sensitive coccobacillus of the family Pasteurellaceae. Strains of the species are currently classified into five serogroups (A, B, D, E, F) based on capsular composition and 16 ...

'' bacterium isoform (pmHAS) contains 972 residues in which deletion of residues 1-117 does not affect enzyme activity, and the

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

of the active site resides around residues 686-703. Two catalytic residues exist in the catalytic domain; an

aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protei ...

asparagine Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...

mutation at position 196 (D196N) leads to loss of GlcUA-

transferase In biochemistry, a transferase is any one of a class of enzymes that catalyse the transfer of specific functional groups (e.g. a methyl or glycosyl group) from one molecule (called the donor) to another (called the acceptor). They are involved ...

activity, and an aspartic acid to

lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. Lysine contains an α-amino group (which is in the protonated form when the lysine is dissolved in water at physiological pH), an α-carboxylic acid group ( ...

mutation at position 477 (D477K) leads to loss of GlcNAc-transferase activity. Combination of both mutants leads to similar activity compared to the wild type. pmHAS additionally contains an Asp-Gly-Ser sequence that is conserved among beta-

glycosyltransferases Glycosyltransferases (GTFs, Gtfs) are enzymes ( EC 2.4) that establish natural glycosidic linkages. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar (also known as the "glycosyl donor") to a nucleophilic glyc ...

. HAS2 is regulated by

dimerization In chemistry, dimerization is the process of joining two identical or similar molecular entities by bonds. The resulting bonds can be either strong or weak. Many symmetrical chemical species are described as dimers, even when the monomer is u ...

and

ubiquitination Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 19 ...

. In COS-1 monkey kidney cells transfected with mouse HAS2 and HAS3

plasmids A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

, one site of ubiquitination is seen on the

at residue 190. A K190R mutant formed a HAS2 dimer, and a flag-tagged and 6myc-tagged HAS2 and HAS3 showed the formation of both homo and

heterodimers In biochemistry, a protein dimer is a macromolecular complex or multimer formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ...

with each other. ''

Chlorella ''Chlorella'' is a genus of about thirteen species of single- celled or colonial green algae of the division Chlorophyta. The cells are spherical in shape, about 2 to 10 μm in diameter, and are without flagella. Their chloroplasts contain t ...

'' virus HAS (Cv-HAS) share roughly 45% sequence similarity to human HAS2. HAS3 is regulated through truncation of the 3’UTR end, which down-regulates

NUDT21 Cleavage and polyadenylation specificity factor subunit 5 (CPSF5) is an enzyme that in humans is encoded by the ''NUDT21'' gene. It belongs to the Nudix family of hydrolases. The protein encoded by this gene is one subunit of the cleavage factor ...

, a regulator for

polyadenylation Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In euka ...

. The loss of this regulatory site in rodent models drives

neoplastic A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...

processes, increased pro-remodeling

phenotypes In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology (physical form and structure), its developmental processes, its biochemical and physiological properti ...

, and the elevation of HA synthesis, but also increases likelihood of

pulmonary hypertension Pulmonary hypertension (PH or PHTN) is a condition of increased blood pressure in the pulmonary artery, arteries of the lungs. Symptoms include dypsnea, shortness of breath, Syncope (medicine), fainting, tiredness, chest pain, pedal edema, swell ...

. HAS3 has a higher sequence identity to HAS2 (71%) compared to HAS1 (57%). HAS1, HAS2, and HAS3 have thirteen, fourteen, and fourteen

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

residues, respectively, compared to '' S. pyogenes'' (spHAS) which contains six cysteine residues. A

sulfhydryl In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...

poisoned cysteine at position 225 in spHAS inhibits enzyme activity, but it is uncertain whether the

disulfide bonds In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In in ...

yield a significant effect on the activity of the HAS enzymes. Hydropathy plots among the three isoforms HAS1, HAS2, and HAS3 suggest that each protein is organized in a similar manner in the membrane.

Mechanism

Initiation of hyaluronan biosynthesis by Class I hyaluronan synthases involves in situ generation of an N-acetylglucosamine (GlcNAc) primer through

hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

of UDP-GlcNAc, followed by diffusion of the primer into the active site. The

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

contains two distinct domains, each of which are capable of binding either the nascent UDP-hyaluronan chain or a UDP-sugar

monomer A monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or two- or three-dimensional network in a process called polymerization. Classification Chemis ...

. Chain elongation, beginning from the GlcNAc primer, occurs with sequential addition of alternating UDP-GlcA and UDP-GlcNAc units to the reducing end of the growing chain. Hyaluronan synthase mechanism

In each iteration of chain elongation, one active site domain is occupied by the existing UDP-hyaluronan chain. A UDP-sugar monomer corresponding to the next unit then binds to the unoccupied active site domain. Subsequently, a

hydroxyl In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...

group on the bound UDP-sugar monomer performs a

nucleophilic attack In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they a ...

on the

anomeric In carbohydrate chemistry, a pair of anomers () is a pair of near-identical stereoisomers or diastereomers that differ at only the anomeric carbon, the carbon atom that bears the aldehyde or ketone functional group in the sugar's open-chain for ...

carbon of the reducing end monomer of the UDP-hyaluronan chain, displacing UDP from the hyaluronan chain and shifting the elongated chain to the domain previously occupied by the UDP-sugar monomer. Following this process, the displaced UDP dissociates from the other active site domain. The process of monomer binding and elongation then repeats, with alternating GlcA and GlcNAc units being added as the UDP-hyaluronan chain shifts from one active site domain to the other. HAS1, HAS2, and HAS3 perform functionally equivalent hyaluronan biosyntheses but demonstrate differences in kinetic characteristics, including HAS1 demonstrating a higher Michaelis constant than HAS2 or HAS3. HAS kinetics and

protein trafficking Protein targeting or protein sorting is the biological mechanism by which proteins are transported to their appropriate destinations within or outside the cell. Proteins can be targeted to the inner space of an organelle, different intracellular m ...

are also influenced by

posttranslational In molecular biology, post-translational modification (PTM) is the covalent process of changing proteins following protein biosynthesis. PTMs may involve enzymes or occur spontaneously. Proteins are created by ribosomes, which translate mRNA int ...

and

epigenetic modifications Embryonic stem cells are capable of self-renewing and differentiating to the desired fate depending on their position in the body. Stem cell homeostasis is maintained through epigenetic mechanisms that are highly dynamic in regulating the chromati ...

Role in cancer metastasis

HAS can play roles in all of the stages of cancer metastasis. By producing anti-adhesive HA, HAS can allow tumor cells to release from the primary tumor mass and if HA associates with receptors such as CD44, the activation of Rho GTPases can promote EMT of the cancer cells. During the processes of intravasation or extravasation, the interaction of HAS produced HA with receptors such as CD44 or RHAMM promote the cell changes that allow for the cancer cells to infiltrate the vascular or lymphatic systems. While traveling in these systems, HA produced by HAS protects the cancer cell from physical damage. Finally, in the formation of a metastatic lesion, HAS produces HA to allow the cancer cell to interact with native cells at the secondary site and to produce a tumor for itself. Increased HA production by cancer cells increases invasive capacity. HA's interaction with CD44 activates focal adhesion kinase (FAK), an important molecule in the process of cell motility by coordinating dissolution of the focal adhesions at the leading edge of the cell and formation at the lagging edge. Another signaling pathway activated by HA's interaction with CD44 is the Akt pathway which leads to expression of osteopontin, a molecule which can stimulate cell migration. The HA produced by HAS also has been suggested to protect the cancer cell from physical damage while in the circulatory or lymphatic systems. This role of HA has been shown in other cell types, but has not yet been researched in cancer cells. The HA produced by HAS up-regulates secretion of various MMPs, proteolytic enzymes that are involved in many stages of the metastatic cascade. Research has shown that the different HASs may impact the metastatic steps in different ways based on the molecular weight and amount of HA they produce.

References

External links

* * {{Portal bar, Biology, border=no EC 2.4.1