Biosynthesis of a tumor cell surface sialomucin. Maturation and effects of monensin.
The major cell surface glycoprotein (ascites sialoglycoprotein-1 (ASGP-1] of ascites 13762 rat mammary tumor cells is a large (Mr greater than 500,000), highly glycosylated sialomucin which is present in great abundance (greater than 0.5% of total cell protein). Thus, these tumors provide a useful system for investigating the biosynthesis of O-glycosylated glycoproteins. Previous studies in this system have demonstrated that initiation of O-linked oligosaccharides occurs throughout most of the transit period of ASGP-1 from the endoplasmic reticulum to the cell surface. By pulse-chase threonine labeling and precipitation with peanut agglutinin, ASGP-1 is first observed as an immature lightly glycosylated form (Mr approximately 200,000) which is converted to a more mature, more heavily glycosylated form (designated the premature or P form) with a half-time of about 30 min. The P form is then more gradually converted into the mature ASGP-1. Analysis of glucosamine-labeled oligosaccharitols obtained from the immature form showed primarily unsialylated derivatives consisting of the structures of the size of the tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc and smaller, whereas the mature form showed a mixture of sialylated and unsialylated structures. Desialylation of glucosamine-labeled mature form resulted in a glycoprotein intermediate in size between the immature and mature forms, indicating that the size change with maturation is not solely due to sialylation. Treatment of the cells with 10(-6) M monensin significantly reduced the conversion of immature to mature form without inhibiting initiation of O-linked oligosaccharides and without preventing sialylation. Analysis of oligosaccharitols obtained from ASGP-1 of monensin-treated cells showed that the major oligosaccharides are trisaccharide GlcNAc beta 1,6(Gal beta 1,3)GalNAc and sialylated trisaccharide GlcNAc beta 1,6(NeuAc alpha 2,3-Gal-beta 1,3) GalNAc. These results suggest that monensin specifically disrupts the compartment of the biosynthetic pathway which adds most of the beta 1,4-Gal to the oligosaccharides of ASGP-1 and that this compartment is separate from the primary site of sialylation.
Spielman
J
J
Department of Anatomy and Cell Biology, University of Miami School of Medicine, Florida 33101.
Hull
S R
SR
Sheng
Z Q
ZQ
Kanterman
R
R
Bright
A
A
Carraway
K L
KL
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D007118:Immunoassay; D037102:Lectins; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology*; D055264:Mucin-4; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis; D019887:Peanut Agglutinin; D051381:Rats; D012795:Sialoglycoproteins / Q000032:analysis / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured
D037102:Lectins; D055264:Mucin-4; D009844:Oligosaccharides; D019887:Peanut Agglutinin; D012795:Sialoglycoproteins; D008985:Monensin; D009439:Neuraminidase