Hyaluronan Matrices in Vascular Pathologies
Shared Resources Core
The goal of our core is to maintain and develop state of the art technologies for glycosaminoglycan (GAG) analysis to serve the life science community in heart, lung and blood research. Our expertise includes all GAGs, with a particular emphasis upon chondroitin sulfate, heparin/heparan sulfate, and hyaluronan.
- Ron Midura, PhD (Core Director)
- (216) 445-3212
- midurar at ccf.org
Overview of Glycosaminoglycan Structure and Function
Glycosaminoglycans (GAGs) are unbranched polysaccharides composed of repeating disaccharide structures that are typically sulfated at specific sites on the backbone. The repeating disaccharide structure of hyaluronan (HA) and heparan sulfate (HS) are the same, with the exception that the Glucuronic acid (GlcUA) and N-Acetylglucosamine (GlcNAc) repeating units are linked GlcUA-β1-3-GlcNAc-β1-4- for HA and GlcA-β1-4-GlcNAc-α1-4- for HS. The repeating disaccharide structure of chondroitin sulfate (CS) is GlcUA and N-Acetylgalactosamine (GalNAc), linked GlcA-β1-3-GalNAc-β1-4-. While HA is never sulfated, the disaccharides of both HS and CS can be unsulfated, or sulfated with 1-3 sulfate groups at specific carbons of both the uronic acid and the sugar amine. Both HS and CS are always attached to a serine residue on core proteins via a xylose-galactose-galactose-GlcUA tetrasaccharide linker. HA is never attached to a core protein. The size of the HS and CS chains are typically 10-30 kDa, while the size of HA is much larger (>1500 kDa). The negative charge of GAGs promotes tissue hydration and their interaction with growth factors and receptors. In particular, the distinct sulfation patterns of CS and HS promote specific protein-GAG interactions that influence a wide range of events during tissue development, homeostasis, and disease.
Our research group at the Cleveland Clinic (including Anthony Calabro, Aimin Wang, Ronald Midura and Vincent Hascall) were the original pioneers who adapted fluorophore-assisted-carbohydrate-electrophoresis (FACE) to the analysis of GAGs (review in Osteoarthritis and Cartilage, vol. 9, Supplement A, pp. S16-S22, 2001). FACE is a rapid, simple and sensitive method for detection and quantification of internal disaccharide and non-reducing termini structures, and of sulfation patterns of chondroitin, dermatan, heparan and keratan sulfate. This technique uses reductive amination to fluorotag GAG disaccharides. Once fluorotagged, the labeled disaccharides are electrophoretically separated on a polyacrylamide gel and visualized on an ultraviolet trans-illuminator. Hyaluronan and chondroitin sulfate disaccharides are analyzed on the same gel, and heparan sulfate, from the same sample, is analyzed on a separate gel. The advantages of this technique include: (1) picomolar sensitivity for GAG detection, (2) the ability to analyze 48 samples in a single 1 hr of electrophoresis, (3) presents individual disaccharides as bands on a gel, making it easy to understand by non-glycobiologists, (4) the bands can be quantified using standard densitometry software, (5) it is relatively simple to learn and (6) it can be used by the broader scientific community without the need to purchase expensive analytical equipment. Click to download our FACE Protocol.
Chondroitin Sulfate Disaccharide Analysis by Fluorophore Assisted Carbohydrate Electrophoresis (FACE). (A & B) FACE gels showing the migration pattern of chondroitin sulfate internal disaccharides with different sulfation patterns. The unique electrophoretic migration of these disaccharides is used to profile chondroitin sulfate disaccharides from unknown samples (not shown). Monosaccharides at non-reducing termini (i.e. 6S-GalNac, 4S-GalNac) can be identified using mercuric ion treatment (not shown). This procedure was developed within our research group at the Cleveland Clinic (Anthony Calabro, Vincent Hascall and Ron Midura; Glycobiology, vol. 10, no. 3 pp. 273-281, 2000). Please see our project 5 for more information on a CS proteoglycan known as “versican.”
Heparan Sulfate Disaccharide Analysis by Fluorophore Assisted Carbohydrate Electrophoresis (FACE). (A & B) FACE gels showing the migration pattern of heparan sulfate disaccharide standards +/- mercuric ion treatment. The unique electrophoretic migration of these disaccharides is used to profile heparan sulfate disaccharides from unknown samples (not shown). Mercuric ion treatment is used to identify monosaccharides at non-reducing termini, removing the Δdi-GlcUA on all internal disaccharides generated by the eliminase mechanism (not shown). Non-reducing ends are not modified. This procedure was developed within our research group at the Cleveland Clinic (Aimin Wang and Vincent Hascall, Osteoarthritis and Cartilage, vol. 9, Supplement A, pp. S16-S22, 2001). Please see our project 1 for more information on the role of heparan sulfate in diabetic vascular pathologies.
Leukocytes Bound to Pathological Hyaluronan Matrices In An Ocluded Pulmonary Blood Vessel from a Patient with Idiopathic Pulmonary Arterial Hypertension
Fluorescent micrograph of a paraffin section probed with the Hyaluronan binding protein (HABP) (green) and antibodies against inter-alpha-inhibitor (red) and the common leukocyte antigen CD435 (magenta). DAPI stained nuclei are shown in blue. The colocalization of Hyaluronan with inter-alpha-inhibitor provides evidence that the Hyaluronan matrices in this micrograph are modified with heavy chains from inter-alpha-inhibitor. This pathological modification enhances leukocyte adhesion to Hyaluronan matrices, as is suggested by the co-localization of leukocytes within this matrix. Mag. 20x