2009 Conference abstract
New Advances in Heparan Sulfate/Heparin Analysis and Biosynthesis
Robert J. Linhardt
Head, Biocatalysis and Metabolic Engineering Constellation, Professor of Chemistry and Chemical Biology, Biology, and Chemical
and Biological Engineering, Rensselaer Polytechnic Institute, Biotech Center 4005, 110 8th Street, Troy, NY 12180-3590, USA.
e-mail: linhar@rpi.edu.
The heparan sulfate/heparin family of glycosaminoglycans and proteoglycans has critical importance in physiology and
pathophysiology as well as being a critical therapeutic agent. Disaccharide analysis is a first step in the analysis of
glycosaminoglycans. Following tissue recovery of glycosaminoglycans or proteoglycans disaccharide composition can now be
definitively determined on as little as 104 cells using liquid chromatography (LC)-mass spectrometry (MS) analysis.
Oligosaccharide mapping affords critical structural information on sequence motifs the size of protein binding sites within
glycosaminoglycans. Sequencing of these oligosaccharides is now possible using Fourier transform ion cyclotron resonance
(FTICR) MS with electron detachment dissociation. MS analysis of full glycosaminoglycan chains, particularly ones with low sulfo
group substitution levels, is now possible. Using preparative gel electrophoresis, the recovery of a single intact
glycosaminoglycan for sequence analysis may now be possible.
Improved analytical technology and a better understanding of glycosaminoglycan and proteoglycan structure should help us
establish structure-activity relationships (SAR). Analysis of the glycosaminoglycan-ome of embryonic stem cells as they
differentiate down different lineages is resulting in important insights into heparan sulfate/heparin SAR. While signaling through
cell surface heparan sulfate is clearly tied its primary structure or sequence, the precise relationship still remains unclear.
An alternative approach to understanding the structure of heparan sulfate/heparin is to better understand heparin biosynthesis
in the Golgi. Two approaches currently underway in our laboratory to explore biosynthesis include the metabolic engineering of
CHO cells to control heparan sulfate/heparin structure and the construction of an artificial Golgi on a digital microfluidic platform.
These approaches should also provide an insight into the control of the outcome of biosynthesis in the Golgi.
Finally, research is underway to develop large-scale enzyme-assisted synthesis of heparan sulfate/heparin. This worked is
directly aimed at replacing animal sourced pharmaceutical heparin with a bioengineered product. This work also offers an
inexpensive approach to the synthesis of heparan sulfate/heparin oligosaccharides. These can also be made with the
incorporation of isotopes, unnatural functional groups or reactive functional groups. These modified heparan sulfate/heparin
oligosaccharides should greatly assist the study of the biology of this important family of molecules.
New Advances in Heparan Sulfate/Heparin Analysis and Biosynthesis
Robert J. Linhardt
Head, Biocatalysis and Metabolic Engineering Constellation, Professor of Chemistry and Chemical Biology, Biology, and Chemical
and Biological Engineering, Rensselaer Polytechnic Institute, Biotech Center 4005, 110 8th Street, Troy, NY 12180-3590, USA.
e-mail: linhar@rpi.edu.
The heparan sulfate/heparin family of glycosaminoglycans and proteoglycans has critical importance in physiology and
pathophysiology as well as being a critical therapeutic agent. Disaccharide analysis is a first step in the analysis of
glycosaminoglycans. Following tissue recovery of glycosaminoglycans or proteoglycans disaccharide composition can now be
definitively determined on as little as 104 cells using liquid chromatography (LC)-mass spectrometry (MS) analysis.
Oligosaccharide mapping affords critical structural information on sequence motifs the size of protein binding sites within
glycosaminoglycans. Sequencing of these oligosaccharides is now possible using Fourier transform ion cyclotron resonance
(FTICR) MS with electron detachment dissociation. MS analysis of full glycosaminoglycan chains, particularly ones with low sulfo
group substitution levels, is now possible. Using preparative gel electrophoresis, the recovery of a single intact
glycosaminoglycan for sequence analysis may now be possible.
Improved analytical technology and a better understanding of glycosaminoglycan and proteoglycan structure should help us
establish structure-activity relationships (SAR). Analysis of the glycosaminoglycan-ome of embryonic stem cells as they
differentiate down different lineages is resulting in important insights into heparan sulfate/heparin SAR. While signaling through
cell surface heparan sulfate is clearly tied its primary structure or sequence, the precise relationship still remains unclear.
An alternative approach to understanding the structure of heparan sulfate/heparin is to better understand heparin biosynthesis
in the Golgi. Two approaches currently underway in our laboratory to explore biosynthesis include the metabolic engineering of
CHO cells to control heparan sulfate/heparin structure and the construction of an artificial Golgi on a digital microfluidic platform.
These approaches should also provide an insight into the control of the outcome of biosynthesis in the Golgi.
Finally, research is underway to develop large-scale enzyme-assisted synthesis of heparan sulfate/heparin. This worked is
directly aimed at replacing animal sourced pharmaceutical heparin with a bioengineered product. This work also offers an
inexpensive approach to the synthesis of heparan sulfate/heparin oligosaccharides. These can also be made with the
incorporation of isotopes, unnatural functional groups or reactive functional groups. These modified heparan sulfate/heparin
oligosaccharides should greatly assist the study of the biology of this important family of molecules.
Research authored by Dr. Linhardt
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List of Publications via PubMed
(NIH National Library of Medicine)
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List of Publications via PubMed
(NIH National Library of Medicine)
| Robert J. Linhardt, Ph.D., research |
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