Purification and characterization of human lysosomal membrane glycoproteins.
Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.
Mane
S M
SM
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Marzella
L
L
Bainton
D F
DF
Holt
V K
VK
Cha
Y
Y
Hildreth
J E
JE
August
J T
JT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007425:Intracellular Membranes / Q000032:analysis* / Q000648:ultrastructure; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000032:analysis* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins