Structure/Property Relationship Characterization
The first step in the modulation of cellular activity by carbohydrates is the recognition and binding of the polymer by pattern recognition receptors located on cell membranes. Glucans and mannans, produced by a wide range of fungi, exhibit different structures in the cell wall of different fungi, which in turn can result in different and unique biological responses as they interact with glucan- or mannan-specific cell surface receptors. In addition, the process of isolating the glucan or mannan can dramatically change the structure. So the structure of the isolated carbohydrate used in medical applications may be considerably different from the structure in the cell wall and thus exhibit considerably different properties than those from the intact cell wall. It is important to recognize that several factors including the fungal growth conditions, isolation protocols and fungal species and their mutants can influence the structure and properties of the carbohydrate polymer. Therefore, it is critical to be able to relate the structure of the isolated carbohydrate to its biological and medical properties as well as desired biological and medical applications based on a solid foundation in structure/property relationships.
Structure/property relationship is greatly aided by characterization of the carbohydrates using well-established structural and physico-chemical analysis techniques. These techniques enable examination of many important structural and physico-chemical properties for these isolates to best determine how they will interact with the desired biological system as well as assist in maintaining product quality of the isolated carbohydrate in the production environment. Structural and physico-chemical properties of greatest interest include the following: the structure of the carbohydrate’s main chain and side chains, its solution conformation, degree or frequency of side chain branches, location of the side chain branch points along the carbohydrate polymer backbone, length of the side chains, molecular weight, polydispersity, degree of polymerization, the nature of the monomers present along with their substitution patterns, monomer linkage types, carbohydrate purity and ligand-receptor binding (1).
AppRidge International, LLC, offers Nuclear Magnetic Resonance Spectroscopy (NMR), multi-detector Gel Permeation Chromatography using Multi-Angle Laser Light Scattering (GPC/MALLS), differential viscometry (GPC/DV) and refractive index detection systems, Gas Chromatography with Mass Spectrometry detection (GC-MS), Electrospray Mass Spectrometry (ES-MS), Matrix Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and Surface Plasmon Resonance Spectroscopy (SPR) in addition to other techniques for complete structure/property relationship analysis of fungal cell wall carbohydrates, such as glucans and mannans, as well as other carbohydrate polymers.
NMR is the most powerful analytical technique available today for the structural characterization of complex carbohydrates. 1D and 2D proton and carbon-13 NMR have proven to be very useful for characterizing structural features of mannans as well as water-soluble and water-insoluble (particulate) glucans isolated from many fungal sources. Proton NMR provides a means to characterize carbohydrates in terms of their purity, monomer types, backbone chain linkage types, side chain linkage types, average side chain lengths, average side chain frequency along the polymer backbone and side chain placement along the backbone or on the reducing terminus as well as impurity content in terms of other carbohydrate polymers and monomers, lipids and fatty acids, proteins, identifiable isolation process residues and sometimes the polymer molecular weight. It may be possible to provide structural characterization of these carbohydrates across the entire spectrum of purity from isolates of high purity to low purity in the presence of a complex residual biomatrix.
GPC/MALLS and GPC/DV provide rapid, simple and accurate determination of a number of important physico-chemical carbohydrate properties including weight-average molecular weight down to about 7,000 kDa, polydispersity, root mean square radius and solution conformation. Molecular weights below 7000 kDa may be analyzed by ES-MS and MALDI-TOF-MS.
GC/MS with permethylated alditol derivatization chemistry enables quantification and identification of linkage types, monomer types and monomer substitution patterns. Careful enzymatic degradation of the polymer chain and its side chains coupled with GC/MS enables examination of the backbone and side chain linkages, lengths and compositions as well as branchpoint characterization.
Surface Plasmon Resonance Spectroscopy (SPR) characterizes binding between carbohydrate ligands and receptors. Polymer structure, molecular weight and solution conformation greatly impact the receptor binding affinity for carbohydrates such as mannans and glucans.
Taken together, results from these techniques enable the development of a structure/property relationship map for these glucans and mannans. These data are important not only for establishing the specific characteristics of a given carbohydrate polymer system, but they are also critical to understanding how the carbohydrate interacts with biological systems. The importance of understanding the structure of carbohydrate polymers, such as glucans and mannans, as a prerequisite for deciphering their biology cannot be overemphasized.
1. “Introduction to the Chemistry and Immunobiology of Beta-Glucans,” David L. Williams, Douglas W. Lowman and Harry E. Ensley, Chapter 1 in “Toxicology of Glucans,” Academic Press, pg. 1-34 (2005)