Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor.
Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.
Altmann
S W
SW
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104.
Prystowsky
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002460:Cell Line; D005822:Genetic Vectors; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D001216:Asparagine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor