Protein and carbohydrate structural analysis of a recombinant soluble CD4 receptor by mass spectrometry.
The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.
Carr
S A
SA
Department of Physical and Structural Chemistry, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
Hemling
M E
ME
Folena-Wasserman
G
G
Sweet
R W
RW
Anumula
K
K
Barr
J R
JR
Huddleston
M J
MJ
Taylor
P
P
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics / Q000302:isolation & purification; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D015870:Gene Expression; D005796:Genes; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011994:Recombinant Proteins
D015704:CD4 Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins