Biochemical Analysis of Snail Secretions

Biochemical analysis of the snail's fluid substance we collect, shows it contains complex glycoconjugates, such as glycosaminoglycans and proteoglycans. These are molecules made mainly of sulfated sugar or carbohydrate chains (sugar= glyco), globular soluble proteins, uronic acids and oligoelements ( copper, zinc, calcium and iron ).

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

Proteoglycans are complex macromolecules consisting of a core protein and one or more covalently attached glycosaminoglycan chains. The biological functions of proteoglycans primarily result 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 polysaccharides. GAGs are usually found in the extracellular matrix of vertebrate and invertebrate tissues. A structural investigation revealed that GAGs in invertebrate species often contain unusual variations of sulfate distribution and uronic acids.

The major glycoconjugate of snail mucous is a glycosaminoglycan, with a novel structure when compared to other known glycosaminoglycans, 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?
They are carbohydrates and are the often-overlooked third major class of biological polymers. Though they have received much less notice than nucleic acids or proteins, they are just as essential for life.

Glycosaminoglycans (GAGs) or mucopolysaccharides are long unbranched polysaccharides, made up of repeating disaccharides that may be sulphated (e.g. glucuronic acid, iduronic acid, galactose, galactosamine, glucosamine).

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 preventing many diseases, glycans appear to act as scaffolds that mediate interactions between proteins.

Carbohydrates are indispensable to life on Earth. In their simplest form, they serve as a primary energy source for sustaining 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.

The Sweet Science of Glycobiology
Complex carbohydrates, molecules that are particularly important for communication among cells, are coming under systematic study. Ram Sasisekharan and James R. Myette See: Glycobiology

The central paradigm of modern molecular biology is that biological information flows from DNA to RNA to protein. The power of this concept lies not only in its template-driven precision, but also in the ability to manipulate any one class of molecules based on knowledge of another, and in the patterns of sequence homology and relatedness that predict function and reveal evolutionary relationships. With the upcoming completion of the genomic sequences of humans and several other commonly studied model organisms, even more spectacular gains in the understanding of biological systems are anticipated. However, there is often a tendency to assume the following extension of the central paradigm:

In actual fact, creating a cell requires two other major classes of molecules: lipids and carbohydrates. These molecules can serve as intermediates in generating energy, as signaling molecules, or as structural components. The structural roles of carbohydrates become particularly important in constructing complex multicellular organs and organisms, which requires interactions of cells with one another and with the surrounding matrix. Indeed, all cells and many macromolecules in nature carry a dense and complex array of covalently attached sugar chains (called oligosaccharides or glycans).

In some instances, these glycans can also be free-standing entities. Since most glycans are on the outer surface of cellular 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 function of a complex multicellular organism. They are also in a position to mediate interactions between organisms (e.g., between host and parasite).

In addition, simple, highly dynamic protein-bound glycans are abundant in the nucleus and cytoplasm, where they appear to serve as regulatory switches. An extended paradigm of molecular biology can thus be rendered as follows:

In the first part of this century, the chemistry, biochemistry, and biology of carbohydrates were very prominent matters of interest. However, during the initial phase of the modern revolution in molecular biology, studies of glycans lagged far behind those of other major classes of molecules. This was in large part due to their inherent structural complexity, the difficulty in easily determining their sequence, and the fact that their biosynthesis could not be directly predicted from the DNA template.

The development of a variety of new technologies for exploring the structures of these sugar chains has opened up a new frontier of molecular biology which has been called glycobiology. This word was first coined in 1988 by Rademacher, Parekh, and Dwek to recognize the coming together of the traditional disciplines of carbohydrate chemistry and biochemistry with modern understanding of the cellular and molecular biology of glycans. The term glycobiology has gained wide acceptance, with a major biomedical journal, a growing scientific society, and a Gordon Research Conference now bearing this name.

Defined in the broadest sense, glycobiology is then the study of the structure, biosynthesis, and biology of saccharides (sugar chains or glycans) that are widely distributed in nature. It is one of the more rapidly growing fields in the biomedical sciences, with relevance to basic research, biomedicine, and biotechnology. Indeed, several biotechnology, pharmaceutical, and laboratory supply companies have invested heavily in the area.

The field ranges from the chemistry of carbohydrates and the enzymology of glycan-modifying proteins to the functions of glycans in complex biological systems, and their manipulation by a variety of techniques. Research in glycobiology requires a foundation not only in the nomenclature, biosynthesis, structure, chemical synthesis, and functions of complex glycans, but also in the general disciplines of molecular genetics, cellular biology, physiology, and protein chemistry. This volume provides an overview of the field of glycobiology, with a particular emphasis on the glycans of higher animal systems, about which the greatest amount is currently known. It is assumed that the reader has a basic background in graduate-level chemistry, biochemistry, and cell biology.

In recent years, important studies of a class of linear glycans known 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 s good deal of light on the role of the snail secretions we use to make the snail cream.

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.

Cell metabolism & the proteins enzymes in the snail's substance speed scar removal and encourage regeneration of skin.