Proteoglycans: Biological Roles and Strategies
351
4.1
Metabolic Labelling of Proteoglycans in Cell Cultures
When the system under consideration is focused on cell cultures, radiolabelling
is the commonest and most convenient way for screening PGs through the various purification steps and measuring recoveries at every purification step. Selective labelling of PGs can be performed by eSSl-sulfate. A high portion of radiosulfate (> 90 %) is incorporated to GAG chains. The carbohydrate precursor,
[3Hl-glucosamine, is also frequently used together with radiosulfate to label
covalently bound GAGs and oligo saccharides to the protein core. Glucosamine
precursor is also of great value for evaluating HA synthesis by the cells and for
subsequent steps, such as the determination of HA. Most cells that synthesize HA
are able to synthesize CSIDSPG, and, therefore, combination of radiosulfate and
[3Hl-glucosamine results in double labelling of CSIDS chains. [2-3Hl-mannose is
another specific carbohydrate precursor useful in evaluating synthesis of Nlinked oligosaccharides. Carbohydrate precursor, generally, can provide useful
information for composition, structure and type of the carbohydrate moieties
bound to the protein core.
Amino acid precursors can be used to label the protein core of PGs as well as
other proteins/glycoproteins. [3Hl-serine and [3Hl-Ieucine are the most common
labels for this purpose and can be used with [ 3s S1-sulfate. Labelling with amino
acids and radiosulfate is useful for estimating the number of different protein
cores in each PG population. Conversion of eHl-serine to alanine following alkaline borohydride treatment (Carlsson 1968) may also be used for determining the
proportion of substituted serine residues of the core protein with GAG chains
and O-linked oligosaccharides. Typical radioisotope concentration should be
within the range from 50 to 250 !lCi/ml of culture. To ensure that synthesized
GAG chains are not under-sulfated eSSl-sulfate concentration in the culture
medium should be at least 0.1 mM. Incorporation of sulfate by cells is linear for
24-48 h following a short (often a few minutes) equilibration time between the
medium and the intracellular precursor pool. Following this labelling procedure
with sulfate, carbohydrate and amino acid precursor all PG and protein/glycoprotein populations present in the medium and the cell-matrix compartments
are labelled.
4.2
Extraction/Solubilization of Proteoglycans
The most effective solutions contain both chaotropic reagents and detergents.
The former will ensure dissociation of non-covalent interactions and denaturation of proteins, whereas the latter dissociation of hydrophobic interactions.
Guanidine hydrochloride (GdnHCl), at a final concentration of 4 M, is the most
effective chaotropic reagent for PGs extraction (Hascall and Kimura 1982). Compatible reagents with guanidine are Triton X-IOO (1-2 % w/v) and CHAPS (2-4 %
w/v). The latter detergent is more convenient since it can be removed by dialysis.
When Triton X-IOO is used, however, care should be taken to avoid formation of
non dialyzable micelles. For extraction of cell bound PGs guanidine should be
351
4.1
Metabolic Labelling of Proteoglycans in Cell Cultures
When the system under consideration is focused on cell cultures, radiolabelling
is the commonest and most convenient way for screening PGs through the various purification steps and measuring recoveries at every purification step. Selective labelling of PGs can be performed by eSSl-sulfate. A high portion of radiosulfate (> 90 %) is incorporated to GAG chains. The carbohydrate precursor,
[3Hl-glucosamine, is also frequently used together with radiosulfate to label
covalently bound GAGs and oligo saccharides to the protein core. Glucosamine
precursor is also of great value for evaluating HA synthesis by the cells and for
subsequent steps, such as the determination of HA. Most cells that synthesize HA
are able to synthesize CSIDSPG, and, therefore, combination of radiosulfate and
[3Hl-glucosamine results in double labelling of CSIDS chains. [2-3Hl-mannose is
another specific carbohydrate precursor useful in evaluating synthesis of Nlinked oligosaccharides. Carbohydrate precursor, generally, can provide useful
information for composition, structure and type of the carbohydrate moieties
bound to the protein core.
Amino acid precursors can be used to label the protein core of PGs as well as
other proteins/glycoproteins. [3Hl-serine and [3Hl-Ieucine are the most common
labels for this purpose and can be used with [ 3s S1-sulfate. Labelling with amino
acids and radiosulfate is useful for estimating the number of different protein
cores in each PG population. Conversion of eHl-serine to alanine following alkaline borohydride treatment (Carlsson 1968) may also be used for determining the
proportion of substituted serine residues of the core protein with GAG chains
and O-linked oligosaccharides. Typical radioisotope concentration should be
within the range from 50 to 250 !lCi/ml of culture. To ensure that synthesized
GAG chains are not under-sulfated eSSl-sulfate concentration in the culture
medium should be at least 0.1 mM. Incorporation of sulfate by cells is linear for
24-48 h following a short (often a few minutes) equilibration time between the
medium and the intracellular precursor pool. Following this labelling procedure
with sulfate, carbohydrate and amino acid precursor all PG and protein/glycoprotein populations present in the medium and the cell-matrix compartments
are labelled.
4.2
Extraction/Solubilization of Proteoglycans
The most effective solutions contain both chaotropic reagents and detergents.
The former will ensure dissociation of non-covalent interactions and denaturation of proteins, whereas the latter dissociation of hydrophobic interactions.
Guanidine hydrochloride (GdnHCl), at a final concentration of 4 M, is the most
effective chaotropic reagent for PGs extraction (Hascall and Kimura 1982). Compatible reagents with guanidine are Triton X-IOO (1-2 % w/v) and CHAPS (2-4 %
w/v). The latter detergent is more convenient since it can be removed by dialysis.
When Triton X-IOO is used, however, care should be taken to avoid formation of
non dialyzable micelles. For extraction of cell bound PGs guanidine should be