Proteolytic Cleavage of Human Growth Hormone (hGH) by Rat Tissuesin Vitro: Influence on the Kinetics of Exogenously Administered hGH*

Abstract

The presence of several endogenous molecular forms of human GH (hGH), including proteolytically cleaved two-chain forms, has been proposed to be related to the diverse biological activity of hGH. The present study characterized hGH degradation in the rat to determine how peripheral metabolism may influence the kinetics and pharmacology of exogenously administered hGH. In vitro studies indicated that hGH was proteolytically degraded by thyroid gland and skeletal muscle, but not liver and kidney homogenates. The proteolytic activity, localized to the 9000 x g pellet fraction, was characterized as a chymotrypsin-like serine protease using class-specific inhibitors. N-Terminal sequencing of hGH peptides formed by the thyroid gland and skeletal muscle indicated that cleavage sites were almost exclusively at Tyr/Phe-Xaa bonds, with similar points of cleavage observed in the two tissues. Immunoreactive two-chain forms of hGH were also formed. The two-chain molecules had similar cleavage sites, but differed in apparent mol wt when analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To understand the potential significance of two-chain product formation, we compared the kinetics and degradation of hGH with those of a synthetic two-chain derivative of hGH (Des-1-8,135-145; 2-CAP). The in vitro tissue distribution of 2-CAP proteolysis was the same as that for hGH. The fragmentation pattern of 2-CAP was less complex when analyzed by reverse phase HPLC. The major peptide fragments formed from 2-CAP were chromatographically similar to those formed from hGH. The plasma kinetics of 2-CAP were compared to those of hGH with a RIA using polyclonal antiserum to hGH. After im and sc administration of 2-CAP (125 micrograms/kg), the area under the plasma concentration curve was 3.2- and 4.5-fold greater, respectively, than after administration of hGH (125 micrograms/kg). Both compounds had a greater area under the curve by the im than the sc route. 2-CAP had 2- to 3-fold greater bioavailability than hGH by the im and sc routes. Plasma from rats treated 30 min earlier with hGH im was immunoextracted and analyzed by Western blotting. A circulating immunoreactive fragment was detected which had similar electrophoretic mobility as a two-chain hGH product formed during the in vitro incubations of hGH with skeletal muscle and thyroid gland homogenates. The results indicate that hGH is proteolytically processed in peripheral tissue homogenates, with the formation of two-chain products. The greater bioavailability of 2-CAP suggests that metabolism of hGH to two-chain forms may influence the in vivo kinetics of hGH.

Host limits to accurate human growth hormone production in multiple plant systems

“…In E. coli, periplasmic secretion can lead to a single cleavage after Thr142 (Canova-Davis et al, 1990). In both humans and other mammalian systems, cleavages are seen from Arg134 to Lys145, although some of this cleavage may occur during purification (Baumann, 1991;Wroblewski et al, 1991). The data in maize may indicate an initial cleavage in planta, followed by additional exoproteolytic activity, possibly ex planta.…”

Thyroid glands: novel sites of growth hormone action

“…Moreover, the affinity constant of these binding sites (29 nmol/1) is in the same range as the GH concentrations in 4-week-old chickens; thus these binding sites may indeed be mediating a physiological role for GH in T4 production. Alternatively, the reduced affinity of these binding sites may be indicative of a mixed population of GH receptors and/or binding proteins and enzymes, as the thyroid gland contains chymotrypsin-like serine pro¬ teases that specifically cleave GH (Wroblewski et al 1991) and it is generally considered that enzymes bind substances with a lower affinity than do true receptors.…”

Pharmacokinetics, Metabolic Stability, and Subcutaneous Bioavailability of a Genetically Engineered Analog of DcR3, FLINT [DcR3(R218Q)], in Cynomolgus Monkeys and Mice

“…DcR3 is also extensively degraded upon s.c. administration, but we were able to identify a highly sensitive cleavage site that appears to be a major initial event in the degradation of this molecule, at least when injected into the subcutaneous space. Characterizing the mechanism of the limited proteolysis of DcR3 involved the application of a number of analytical approaches along with a correlation of data obtained in vivo with that from in vitro models (Wroblewski et al, 1991(Wroblewski et al, , 1993a(Wroblewski et al, , 2003. Physiologically, limited proteolysis may lead to inactivation of the parent protein, but has been shown to yield variants having altered receptor affinities and enhanced or selective biological activities (Korc and Finman, 1989;Powers and Hatala, 1990;Wroblewski et al, 1993b;Angelloz-Nicoud et al, 1998;Bergsten et al, 2001;Sharpe and DeMeester, 2001).…”