Student Theses and Dissertations

Date of Award

1983

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Bruce Cunningham

Keywords

L-CAM, cell adhesion, embryogenesis, glycoproteins, monoclonal antibodies, epithelial cells

Abstract

Cell-cell adhesion is one of the essential processes for normal embryogenesis. The isolation and characterization of the neural cell adhesion molecule, N-CAM, provided the first substantial opportunity to study this process at a molecular level. Moreover, studies of N-CAM expression in embryos suggested that cell adhesion molecules may play a critical role in induction. This thesis describes the isolation of a glycoprotein, L-CAM, that mediates adhesion between liver cells, using an assay similar to the one used to isolate N-CAM. L-CAM was released from liver cell membranes by proteolysis with trypsin, and purified by ion-exchange chromatography, gel filtration chromatography and isoelectric focusing. The purified L-CAM was used to produce monospecific antibodies in rabbits and monoclonal antibodies from murine hybridomas. The identity of the L-CAM activity with the isolated molecule was confirmed using these immunological reagents, by demonstrating their capacity for disrupting cell adhesion and by showing that these antibodies were neutralized by the purified L-CAM molecule. L-CAM is an acidic intrinsic membrane glycoprotein of Mr=124,OOO daltons. It is approximately 12% neutral sugar, with a small but demonstrable amount of sialic acid. The carbohydrate is distributed as four approximately equal sized asparagine-linked oligosaccharide chains. Only one of the oligosaccharide chains is susceptible to cleavage by endoglycosidase H, suggesting that it is of the high-mannose type; the other three are probably of the complex type. Antibodies to L-CAM inhibit calcium-dependent cell-cell adhesion between liver cells in a rotary suspension culture. The same antibodies inhibit the formation of histotypic liver cell colonies that form when dissociated liver cells are cultured on a solid substrate. Colonies that have already formed can be disrupted by the antibodies to L-CAM. These histotypic colonies have some physiological properties comparable to intact livers. These results suggest that it is possible to investigate the role of cell-cell contact in some of the physiological functions of the liver using anti-L-CAM as a perturbing agent. N-CAM and L-CAM are two different molecules, mediating different processes of cell adhesion. L-CAM, however, does bear striking structural and functional homologies to a molecule (uvomorulin) that mediates adhesion between cells in very early embryos. This similarity, considered with the known appearance of N-CAM in very early embryos, suggests that N-CAM and L-CAM may play complementary roles in the processes of early embryogenesis. In accord with this hypothesis, LCAM was found to be widely distributed in organs of embryos and adult animals. L-CAM was found on organs that arise from all three germ layers; in the organs where it was detected, the molecule was restricted to epithelial cells, and in some cases to only part of the cell surface. This result provides support for Edelman's hypothesis that a few cell-adhesion molecules can play major roles in a variety of developing organs and tissues. L-CAM, along with N-CAM, appears to be among these cell adhesion molecules.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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