Studies on the Asparagine-Linked Oligosaccharides From Cartilage-Specific Proteoglycan (Chondrocytes)
Cioffi, Linda Carol
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https://hdl.handle.net/2142/70565
Description
Title
Studies on the Asparagine-Linked Oligosaccharides From Cartilage-Specific Proteoglycan (Chondrocytes)
Author(s)
Cioffi, Linda Carol
Issue Date
1987
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Chondrocytes synthesize and secrete a cartilage-specific proteoglycan (PG-H) as one of their major products. This proteoglycan has attached to it several types of carbohydrate chains, including chondroitin sulfate, keratan sulfate, O-linked oligosaccharides, and asparagine-linked oligosaccharides. The asparagine-linked oligosaccharides found on PG-H were investigated in these studies.
Methodology was developed for the isolation and separation of standard of standard complex and high mannose type oligosaccharides. This included digesting glycoproteins with N-glycanase and separation of the oligosaccharides according to type by concanavalin-A lectin chromatography. The different oligosaccharide types were then analyzed by high pressure liquid chromatography. This methodology was used in the subsequent studies on the PG-H asparagine-linked oligosaccharides.
Initially, the asparagine-linked oligosaccharides recovered from the culture medium (CM) and cell-associated (Ma) fractions of PG-H from of tibial chondrocytes were labeled with ($\sp3{\rm H}$) -mannose and the oligosaccharides were isolated and analyzed. The asparagine-linked oligosaccharides were found to be heterogeneous mixtures of complex and high mannose structures in both the CM and Ma PG-H pools. The ratio of complex to high mannose type oligosaccharides was found to be 6.1 in the CM PG-H and 2.6 in the Ma PG-H. These results suggest the possibility that the CM and Ma PG-H are two separate and distinct molecules, each having its own structure and function.
The chick embryo tibiotarsus was then dissected into zones which differed in their states of differentiation. The PG-H from monolayer cultures of these distinct zones of chondrocytes were isolated and the asparagine-linked oligosaccharides from the CM and Ma pools were characterized are found to be heterogeneous mixtures of oligosaccharide structures. The PG-H from each zone gave its own characteristic array of structures.
In addition to cell culture studies, the asparagine-linked oligosaccharide structures from PG-H in tissue culture were investigated. In every system analyzed--mixed zone cell culture, distinct zone cell culture, mixed zone embryo tibial tissue, and the distinct zones of the chick embryo tibial tissue--the asparagine-linked oligosaccharides were found to be a heterogeneous mixture of complex and high mannose structures.
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