Recombinant human insulin-like growth factor binding proteins 4, 5, and 6: biological and physiochemical characterization.
We have recently cloned cDNAs encoding human insulin-like growth factor binding proteins (IGFBP)-4, -5 and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins. Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating expression and processing of the fusion proteins. HPLC purified rhIGFBPs had virtually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs. Rabbit antiserum directed against each rhIGFBP-4, -5 and -6 reacted specifically with the respective rhIGFBP as well as with the native human counterpart and displayed very low cross-reactivity with other IGFBPs. Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1. When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, although rh-IGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant.
Kiefer
M C
MC
Chiron Corporation, Emeryville, CA 94608.
Schmid
C
C
Waldvogel
M
M
Schläpfer
I
I
Futo
E
E
Masiarz
F R
FR
Green
K
K
Barr
P J
PJ
Zapf
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D003429:Cross Reactions; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D018976:Insulin-Like Growth Factor Binding Protein 6; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D018976:Insulin-Like Growth Factor Binding Protein 6; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D004247:DNA