95- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor.
125I-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extent with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25-kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.
Nygren
A
A
Department of Immunology, Karolinska Institutet, Stockholm, Sweden.
Bergman
T
T
Matthews
T
T
Jörnvall
H
H
Wigzell
H
H
D002460:Cell Line; D006678:HIV / Q000378:metabolism*; D015699:HIV Envelope Protein gp120; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D015728:Receptors, HIV; D011991:Receptors, Virus / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D015728:Receptors, HIV; D011991:Receptors, Virus; D012191:Retroviridae Proteins; D014759:Viral Envelope Proteins