Biochemical Analysis of The Biological Ingredient

Biochemical analysis of the fluid substance we collect from snails, shows it contains a complex compound of glycoconjugates, comprising glycosaminoglycans, glycoproteins and proteoglycans. These are complex glyco molecules made of sulfated sugar or carbohydrate chains (sugar= glyco), globular soluble proteins, uronic acids and several oligoelements ( copper, zinc, calcium and iron ).

Proteoglycans, Glycoproteins and Glycosaminoglycans are active regulators of the cell's functions. They participate in cell-matrix interactions and play an important biological role in fibroblasts proliferation, and in the differentiation and migration of cells by effectively modulating the cellular phenotype .

Proteoglycans are complex macromolecules consisting of a core protein and one or more covalently attached glycosaminoglycan chain. The biological functions of proteoglycans result primarely from the structurally dominant glycosaminoglycans emanating from the protein core of the molecule. A large number of animal species contain GAGs and mollusks are a particularly rich source of these glycomolecules or polysaccharides.

GAGs are usually found in the extracellular matrix of vertebrate and invertebrate tissues. Structural analysis reveals that GAGs in invertebrate species often contain unusual variations of sulfate distribution and uronic acids.

The major glycoconjugate of the snail fluid is a glycosaminoglycan, with a novel structure when compared to other known glycosaminoglycans. It is secreted from granules within the snail's body and is localized on the outer surface as a result of exposure of the snail to stress.

What are glycosaminoglycans?
Carbohydrates are indispensable to life. In their simplest form, they serve as a primary energy source that sustains life. For the most part, however, carbohydrates exist not as simple sugars but as complex molecular conjugates, or glycans.

Glycans come in many shapes and sizes, from linear chains (polysaccharides) to highly branched molecules bristling with antennae-like arms. And although proteins and nucleic acids such as DNA have traditionally attracted far more scientific attention, glycans are also key to life. They are ubiquitous in nature, forming the intricate sugar coat that surrounds the cells of virtually every organism and occupying the spaces between these cells. As part of this so-called extracellular matrix, glycans, with their diverse chemical structures, play a crucial role in transmitting important biochemical signals into and between cells. In this way, these sugars guide the cellular communication that is essential for normal cell and tissue development and physiological function.

GAGs form an important component of connective tissues. GAG chains may be covalently linked to a protein to form proteoglycans .

Dermatan sulfate is a glycosaminoglycan found mostly in skin, but also in blood vessels, heart valves, tendons, and lungs. Dermatan sulfate may have roles in coagulation, cardiovascular disease, carcinogenesis, infection, wound repair, and fibrosis.

Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars (N-acetyl-galactosamine and glucuronic acid). It is usually found attached to proteins as part of a proteoglycan. A chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities. Understanding the functions of such diversity in chondroitin sulfate and related glycosaminoglycans is a major goal of glycobiology. Chondroitin sulfate is a major structural component of cartilage and provides much of its resistance to compression.

Complex sugars, or glycans, which are generally bound to proteins, coat the outsides of cells and fill the spaces between them. Crucial in normal animal development and in the prevention ofmany diseases, glycans appear to act as scaffolds that mediate interactions between proteins.

The Sweet Science of Glycobiology
The Sweet Science of Glycobiology. Heterogeneus carbohydrates, molecules that are particularly relevant for communication among cells, are coming under systematic study. Glycobiology (Ram Sasisekharan and James R. Myette)

The most important criterion of modern molecular biology is that biological data goes from DNA to RNA to protein. The power of this concept resides not just in its template-driven precision, but also in the capacity to manipulate any one type of molecules based on knowledge of another, and in the diagrams of sequence homology and relatedness that predict activity and show evolutionary connections. With the upcoming finalization of the genomic sequences of humans and several other commonly studied model beings, even more amazing gains in the understanding of biological elements are looked forward to. However, there is commonly an inclination to assume the following extension of the main criterion: DNA to RNA to PROTEIN to CELL to ORGANISM.

In actual fact, elaborating a cell needs two other major classes of molecules: lipids and carbohydrates. These molecules can act as intermediates in generating energy, as signaling molecules, or as structural elements. The structural activities of carbohydrates get specially important in creating heterogeneous multicellular organs and organisms, which requires communication of cells with others and with the surrounding matrix. Actually, all cells and several macromolecules in nature carry a compressed and intrincated array of covalently united sugar chains (called oligosaccharides or glycans).

In some instances, these glycans can equally be free-standing entities. Since the big majority of glycans are on the external surface of cells and secreted macromolecules, they are in a position to modulate or mediate a wide variety of events in cell-cell and cell-matrix interactions crucial to the development and action of an heterogeneous multicellular organism. They are also in a place to control interactions between organisms (e.g., between host and parasite).

Plus, simple, highly dynamic protein-bound glycans are abundant in the nucleus and cytoplasm, where they appear to serve as managing switches.

In the first part of the past century, the chemistry, biochemistry, and biology of carbohydrates were extremely prominent subjects of research. Nevertheless, during the initial years of the modern revolution in molecular biology, reseach about glycans looked small next to those of other major classes of molecules. This was in large part because of their innate structural complexity, the difficulty in easily resolving their sequence, and the fact that their biosynthesis couldn’t be directly infered from the DNA template.

The development of a variety of rather new technologies for exploring the structures of these sugar chains has opened up a new universe of molecular biology which has been named glycobiology. That word was first spoken in 1988 by Rademacher, Parekh, and Dwek to recognize the coming together of the traditional disciplines of carbohydrate chemistry and biochemistry with modern comprehension of the cellular and molecular biology of glycans. The word glycobiology has gainedwide acceptance, with a major biomedical journal, a growing scientific society, and a Gordon Research Conference now using this name.

Explained in the broadest way, glycobiology is the study of the morphology, biosynthesis, and biology of saccharides (sugar chains or glycans) that are widely distributed in nature. It’s one of the most active fields in biomedical sciences, with impact in basic sciences, biomedicine, and biotechnology. Actually, many biotechnology and pharmaceutical enterprises have invested heavily in the field.

The field ranges from the chemistry of carbohydrates and the enzymology of glycan-modifying proteins to the main activities of glycans in intrincated biological systems, and their manipulation by a large variety of techniques. Study in glycobiology requires a foundation not just in the nomenclature, biosynthesis, morphology, chemical synthesis, and activities of intrincated glycans, but also in the common disciplines of molecular genetics, cellular biology, physiology, and protein chemistry.

In these passed years, very important research of a type of linear glycans recognized as glycosaminoglycans (or GAGs for short), and particularly a sub-set known as HSGAGs, which are made up of heparan sulfate and its relative heparin have shed a good deal of light on the role of the snail secretions we use to elaborate our skin care products.

Building the Chains
An HSGAG chain may be generically described as a linear repeat of approximately 10 to 100 disaccharide building blocks that, when linked together, make up the backbone of each sugar molecule. In its most fundamental form, each disaccharide unit consists of two chemically distinct monosaccharides (a uronic acid and a glucosamine) linked by a glycosidic bond. The chains can vary a great deal in their structural configuration because the disaccharide building blocks may be chemically modified at a number of positions.

These modifications include the removal of the two-carbon acetyl groups at the amino position of the glucosamine portion and the addition of sulfate groups at several different positions, along with distinctions in the stereochemical arrangement of bonds around specific carbons. Different combinations of these various chemical modifications make it possible for even short chains to have an enormous number of structural permutations. In fact, the potential for an immense quantity of structural information to be embedded in a glycan exceeds that of nucleic acids or proteins.

Unlike the synthesis of DNA, RNA or proteins, however, glycan synthesis does not depend on a template that codes for the exact sequence of building blocks in a new chain, to be faith-fully replicated over and over again as an identical copy. Instead, GAGs are synthesized through the concerted action of a large repertoire of enzymes whose existence and relative activities vary greatly. In short, HSGAG biosynthesis is a multi-step process with multiple enzyme players.

Most of the enzymes involved in HSGAG biosynthesis are now known, but exactly how the process of synthesis plays out is still very much an open question. We know little about the ratio of enzymes or, even more basically, whether they act independently or co-operatively in a multienzyme complex.

It is known that HSGAGs are made inside the cell in the membranes of the organelles known as the Golgi apparatus. Nearly all the enzymes involved with making HSGAGs either span the organelle's membranes or are at least peripherally associated with them. This arrangement essentially restricts the interaction of these enzymes to two dimensions within a lipid lattice.

Although the complete biochemical picture is not yet known, it is likely that the enzymes for HSGAG biosynthesis come together within the Golgi membrane, perhaps as the chain is being assembled.

For the most part, glycans do not exist at the cell surface or in the extracellular matrix (ECM) as free-standing polymers. Rather, they are assembled onto specific proteins to form protein-glycan conjugates, or proteoglycans. With the exception of heparin, which is made as a free-standing sugar polymer, HSGAGs are generally found in three major classes of proteoglycans.

A major distinction among these proteoglycans may be found in their particular arrangement relative to the cell surface. In syndecans, the core proteins cross the cell membrane. Glypicans are also inserted into membranes, but by a lipid anchor connected to the core protein. Perlecans reside in the ECM. There is much evidence that the particular composition of glycans attached to each core protein is not random.

Structure Determines Function
Proteoglycans are unique and structurally complex macromolecules. A clue to the function of HSGAG proteoglycans comes from the list of important proteins with which they bind in discrete spatial and temporal interactions.

These proteins include many key growth factors and growth-factor receptors, proteins involved in tissue and organ development, others involved in immune and inflammatory responses, some that mediate cell adhesion, and so on. Like proteoglycans, the proteins that associate with them generally reside outside cells, either near cell membranes or dispersed throughout the ECM. Many of these proteins circulate in the blood, where they are involved in processes such as blood coagulation, wound healing and tissue repair.

The interactions between glycans and the proteins they bind to reveal connections between structure and activity. These interactions have often been ascribed merely to the noncovalent electrostatic attraction between negatively charged sugars and positively charged proteins. A closer look, however, reveals that many protein-glycan interactions are in fact structurally selective. We offer three examples of such specific interactions—the binding of HSGAGs to antithrombin, to fibroblast growth factor and to herpes simplex virus gD glycoprotein.

In some cases, exquisite structural specificity guides the interaction between HSGAGs and proteins in a way reminiscent of the so-called lock-and-key complementarity between enzymes and their substrates. The binding of heparin to antithrombin III (or ATIII) is a classic example of such an interaction. ATIII is a protein that plays a key role in the cascade of steps that leads to blood coagulation.

Clinicians have appreciated the influence of heparin on this process since the early 1930s, when heparin was first used as an anticoagulant during surgery. We now know that when heparin binds to ATIII, this binding induces an important change in the conformation of the protein. In turn, this change greatly in-creases the inhibitory action that ATIII exerts on certain other proteins that normally promote blood coagulation. A series of experiments have shown that only a small segment within heparin (which exists as a mixed population of molecules) actually binds to ATIII and induces its conformational change.

The minimal active binding sequence is a distinct pentasaccharide (that is, two-and-a-half disaccharide units). However, to trigger as much anticoagulation as would a full-length heparin molecule, a longer polysaccharide is required, one that can simultaneously bind to the protein thrombin as well as to antithrombin III. Although the HSGAG region that binds to thrombin does not appear to require a precise sequence, its spacing relative to the ATIII-binding region is very important.

This example illustrates two important points about the interaction of proteins with HSGAGs, and probably other glycans. First, the protein-binding region within the polysaccharide is not randomly distributed throughout the chain; rather, it is generally restricted to a limited number of contiguous disaccharides out of the more than 100 that may make up its linear sequence. Second, a single glycan chain often has two or more sites for protein binding. One can think of the glycan, therefore, as a molecular scaffold that promotes the favorable interaction of two or more protein partners.

The example of fibroblast growth factor signaling also elegantly illustrates the concept of HSGAGs bringing proteins together. In particular, the glycans facilitate the interaction of fibro-blast growth factor with its receptor at the cell surface. The binding of growth factor to its receptor sets in motion a signaling cascade that ends up in the cell's nucleus, turning on genes that modulate cellular proliferation. To trigger this cascade, a receptor embedded in the cell membrane needs to undergo a structural change, a change that occurs when one receptor interacts simultaneously with a second receptor.

It seems that the FGF molecules outside the cell (at least in the case of the growth factor known as FGF-2) must themselves form a dimer, or pair, to bring two receptors together on the cell surface. Certain studies have shown that FGF signaling may not absolutely require the presence of the glycan; yet in this convergence of molecules glycans do serve as a sort of glue, holding the entire complex together in the proper configuration necessary for maximal signal transduction.