EVIDENCE THAT LIMITED PROTEOLYSIS OF INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 (IGFBP-3) OCCURS IN THE NORMAL-STATE OUTSIDE OF THE BLOOD-STREAM

In serum and other biological fluids, IGF-I and IGF-II are reversibly bound to six molecular species of specific binding proteins (IGFBP- 1 to -6) which regulate their transport to target cells as well as their biological activities. Most of the IGFs in adult serum are bound to IGFBP-3 and associated with a 85 kDa subunit to form 150 kDa ternary complexes, very few of which cross the capillary barrier. In earlier studies we showed that during pregnancy one or more serine proteinases are responsible for limited proteolysis of IGFBP-3 in the serum. This may result in easier dissociation of the IGFs and hence an increase in their availability. In the present work, the results of Western blot analyses of the IGFBPs, using either labelled IGF or a polyclonal anti-IGFBP-3 antibody, have demonstrated that IGFBP-3 proteolysis occurs in the normal state. In all the adults investigated, serum contained both the 42-39 kDa doublet, characteristic of intact IGFBP-3, and its major degradation fragment of 30 kDa. The fragment was also found in lymph, in addition to smaller fragments of 21.5, 20 and 16 kDa which are the same sizes as those seen in pregnancy serum. Comparisons of lymph (which reflects the interstitium) and serum from the same subjects showed greater proportions of IGFBP-3 proteolysed in lymph than in serum and incubations with [I-125]IGFBP-3 showed almost 8-fold higher proteolytic activity in lymph than in serum where it was minimal. These findings suggest that the initial sites of proteolysis are in the tissues. Like that in pregnancy serum, the activity was calcium-dependent and inhibited by aprotinin. The results of this study fit the hypothesis that limited proteolysis of IGFBP-3 may be an essential mechanism in controlling the bioavailability of the IGFs, both at the cellular level and, in the blood, from the 150 kDa complexes which constitute the circulating reserves of IGF.