Trypsin-resistant forms of human growth hormone have diabetogenic and insulin-like activities

Abstract

Although diabetogenic and insulin-like activities are intrinsic properties of the growth hormone (GH) molecule, it has been frequently suggested that the hormone must be proteolytically processed for these activities to be expressed. If this is correct, then derivatives of GH having resistance to appropriate proteolytic attack might not have diabetogenic and/or insulin-like activity. The purpose of the present study was to prepare derivatives of human GH that are resistant to digestion by trypsin and to determine whether they possess diabetogenic or insulin-like activity. Three derivatives were prepared from purified native human GH in which lysine residues were modified with methyl acetimidate, citraconic anhydride or S-ethyl-thioltrifluoroacetate, and one in which arginine residues were modified with camphorquinone-10-sulfonic acid. Comparisons of peptide maps of tryptic digests of these derivatives with that of unmodified human GH indicated that all four were resistant to proteolysis by trypsin. All of these trypsin-resistant forms of human GH were found to possess significant growth-promoting, diabetogenic and insulin-like activities, although all activities were attenuated to some extent in each derivative. The relative potencies of the human GH derivatives in a radioimmunoassay for human GH were somewhat similar to their order of potency in the growth-promoting and diabetogenic assays. These results suggest that if proteolytic processing of the GH molecule is involved in the expression of one or more of its biological activities, such processing probably does not involve a trypsin-like proteinase.

Biological characterization of purified native 20-kDa human growth hormone

“…4B). This dose response relationship is similar to that routinely obtained in our laboratory with purified native 22-kDa GH [25][26][27]. The in vitro insulin-like activity of the purified native 20-kDa GH preparation is compared with that of native 22-kDa GH in Table V.…”

“…Statistical comparisons were made using the paired t-test, and effects were considered to be significant at the P < 0.05 level. Statistically significant diabetogenic effects are routinely obtained in this assay with 10-25/~g of highly purified native 22-kDa GH [25][26][27].…”

ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

“…After cleavage by reducing agent, half of each crosslinker remains attached to a target residue in the crosslinked protein. In addition, bound crosslinker may alter proteolytic cleavage patterns, as has been demonstrated for other lysine modifications [23]. In addition The cleaved crosslinker alters peptide mass and can prevent recognition by standard MS algorithms [24].…”

Non-classical Signalling of Growth Hormone in the Chick Neural Retina?

“…As GH is lipolytic, another bioassay for chicken GH is its ability to induce free fatty acid release from adipose tissue explants in vitro (Campbell and Scanes, 1985), although anti-lipolytic effects have also been observed (Campbell and Scanes, 1987). Insulin-like effects of ostrich GH on [ 14 C] glucose oxidation by rat adipose tissue (Cameron et al, 1985a) and its diabetogenic effect on in vitro glucose tolerance in ob/ob mice (Cameron et al, 1985b) are other markers of avian GH bioactivity. Full-length chicken GH has also been shown to depress hepatic 5-deiodinase type III activity in vitro and interestingly the 15 kDa fragment was equipotent with recombinant chicken GH in this bioassay (Aramburo et al, 2001).…”