Post-translational processing of membrane-associated neu differentiation factor proisoforms expressed in mammalian cells.
Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.
Lu
H S
HS
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Hara
S
S
Wong
L W
LW
Jones
M D
MD
Katta
V
V
Trail
G
G
Zou
A
A
Brankow
D
D
Cole
S
S
Hu
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020880:Neuregulins; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D006023:Glycoproteins; D020880:Neuregulins; D011994:Recombinant Proteins