Characterisation of glycoprotein II from bovine adrenal medulla

by Hieber, A. David

Abstract (Summary)
Glycoprotein II (GpII) is a glycoprotein isolated from the membranes of chromaffin granules in the adrenal medulla. The chromaffin granules of the adrenal medulla are responsible for the biosynthesis, storage and secretion of catecholamines, neuropeptides and various proteins. The abundance of chromaffin granules makes them an excellent model to further study the organelles specialized in the synthesis and secretion of hormones and neurotransmitters, from both endocrine and synaptic vesicles. When viewed by two-dimensional electrophoresis GpII is a heterogeneoug glycoprotein (80000-100000 daltons) consisting of two components, upper (GpIIa) and lower (GpIIb). Protein and immunological characterisation revealed that the two components of GpII are distinct from each other. Further molecular characterisation of GpIIa showed that it consisted of a 1224 base pair cDNA, encoding a 383 amino acid polypeptide, with a calculated molecular mass of 40500 daltons. This characterisation also revealed 19 potential glycosylation sites, with deglycosylation studies further demonstrating that an estimated 55% of the molecular mass of GpII is asparagine-linked carbohydrate. The presence of poly-N-acetyllactosamine carbohydrate groups on GpII was also demonstrated. The predicted amino acid sequence of GpIIa shares a 72% amino acid identity with the human lamp-l type protein, which belongs to a highly conserved group of lysosomal associated membrane glycoproteins (lamp proteins). As the C-terminal region of GpIIa was identical to the C-terminal region of lamp proteins, a synthetic peptide to the C-terminal region of GpII was used to raise antisera to this region. This antisera was then used to demonstrate that the C-terminal region of GpII wag present on chromaffin granules and facing the cytoplasm. This same C-terminal region on lamp proteins is known to be a cytoplasmic tail that is essential for the intracellular targeting of lamp proteins to the lysosome. A common feature to the pathways of both GpII and lamp proteins is their appearance on the cell surface followed by internalisation via endocytosis. As endocytosis of the chromaffin granule membrane is known to occur, the possibility that GpII was a marker for endocytosis was investigated. The presence of GpII within clathrin coated vesicles was shown, and evidence was presented to demonstrate that the C-terminal region of GpII interacts with adaptor proteins of clathrin coated vesicles. Adaptor proteins are known to mediate between the cytoplasmic domain of proteins internalised by endocytosis, and the outside coat of clathrin. The evidence presented would suggest that the cytoplasmic tail of GpII is a potential marker for endocytosis within the chromaffin granule membrane. The results presented in this study indicate that GpII is the secretory granule and species counterpart to the lamp proteins. This finding however raises some interesting questions regarding intracellular targeting between the lysosomes and secretory granules within the chromaffin cell.
Bibliographical Information:

Advisor:Dr. David Christie

School:The University of Auckland / Te Whare Wananga o Tamaki Makaurau

School Location:New Zealand

Source Type:Master's Thesis

Keywords:fields of research 270000 biological sciences 270100 biochemistry and cell biology


Date of Publication:01/01/1993

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