Disulfide structure and N-glycosylation sites of an extracellular domain of granulocyte-colony stimulating factor receptor.
An extracellular domain containing 603 amino acid residues of human granulocyte-colony stimulating factor receptor was expressed in Chinese hamster ovary cells. The affinity-purified material has previously been shown to dimerize when combined with the ligand. In this paper we have characterized the primary structure of this active receptor. Laser desorption mass spectrometry of the purified receptor showed a broad peak at a molecular weight of 84,000, ranging from 77,000 to 91,000. The molecular weight heterogeneity is due to glycosylation. Since the molecular weight based on the amino acid sequence is 67,322, by subtraction the carbohydrate content is approximately 17,000. Disulfide structure of the receptor was determined by peptide mapping in the absence and presence of reducing agent. Sequence and mass spectral analyses of these peptides showed the receptor to contain eight disulfide bonds and three free cysteines. These disulfide bonds are consistent with the known domain motifs of the receptor in that no interdomain disulfides were present. One of the three free cysteines is reactive with alkylating agents, while the others are less reactive, probably being buried in the interior of the molecule. Blocking the free cysteines did not affect the ligand binding. Carbohydrate moieties are somewhat evenly spaced throughout the molecule, at eight different N-glycosylation sites, some of which show heterogeneity in their compositions. Glycosylation seems necessary for stabilizing the molecule against disulfide-linked oligomerization of the receptor, indicating that the free cysteine residues become reactive for oxidation and disulfide exchange upon deglycosylation.
Haniu
M
M
Department of Protein Chemistry, Amgen, Inc., Thousand Oaks, California 91320, USA.
Horan
T
T
Arakawa
T
T
Le
J
J
Katta
V
V
Hara
S
S
Rohde
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000494:pharmacology; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents; D013820:Thermolysin / Q000378:metabolism
D004220:Disulfides; D010455:Peptides; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D010434:Pepsin A; D013820:Thermolysin; D003545:Cysteine