Generation of 5 and 17 kDa human growth hormone fragments through limited proteolysis GERARD SUCH-SANMARTI ´ N 1 , JAUME BOSCH 1 , JORDI SEGURA 1,2 ,& RICARDO GUTIE ´ RREZ-GALLEGO 1,2 1 Bio-Analysis Group, Neuropsychopharmacology Program, Municipal Institute for Medical Research-Hospital del Mar, Parque de Investigacio ´n Biome ´dica de Barcelona, Barcelona, Spain, and 2 Department of Experimental and Health Sciences, Pompeu Fabra University, Parque de Investigacio ´n Biome ´dica de Barcelona, Barcelona, Spain (Received 27 March 2009; revised 5 June 2009; accepted 10 June 2009) Abstract Introduction. The reported presence of two fragments of 5 and 17 kDa originating from the 22 kDa human growth hormone (hGH) in blood and tissues, postulated as the sequences AA 1–43 and AA 44 – 191 , has led to the hypothesis of a post- translational proteolytic origin with respect to the abundant 22 kDa variant (AA 1 – 191 ). To evaluate this hypothesis, the activity of several endo-proteases on the 22 kDa hGH protein has been evaluated. Methods. Proteolysis using pepsin, trypsin, V8- protease, proteinase K and thermolysin were explored under several conditions, including incubation time and pH. Results were monitored by MALDI-TOF and HPLC-ESI mass spectrometry. Proteolytic 5 and 17 kDa fragments were purified through reversed phase HPLC-UV, and their immuno-affinity properties evaluated by surface plasmon resonance. Results. Thermolysin was shown to target mainly the AA 43 – 44 bond of the 22 kDa sequence at physiological pH. Interaction studies of the purified fragments with anti-GH antibodies showed some reactivity for the 17 kDa fragment. Conclusions. Thermolysin processes hGH generating 5 and 17 kDa fragments, demonstrating the feasibility of this reaction, although the enzyme responsible for this process in humans is still unknown. Specific antibodies should be used to detect these fragments in human specimens, and, at the same time, the 17 kDa fragment could constitute an interference in some hGH immunoassays. Keywords: Human growth hormone fragments, hGH 1–43 , hGH 44 – 191 , limited proteolysis, protein structure, thermolysin Abbreviations: hGH, human growth hormone; HPLC-ESI, high-performance liquid chromatography-electrospray ionisation; SPR, surface plasmon resonance; MALDI-TOF, matrix-assisted laser desorption ionisation-time of flight; MS, mass spectrometry; PMF, peptide mass fingerprint; E/S, enzyme–substrate ratio; RU, resonance units; mAb, monoclonal antibody Introduction Human growth hormone (hGH) represents an exquisite example of biological diversity. At least six different molecular-weight splice variants [22 kDa (Baumann 1999), 20 kDa (Tsushima et al. 1999), 17.5 kDa (Lecomte et al. 1987), 17.8 and 17 kDa (Zhan et al. 2005) and 11.3 kDa (Palmetshofer et al. 1995)] have been described, and several post- translational point mutations occur. In circulation, where all variants may exist as monomers, homo/ hetero di- and oligomers, either alone or in complex with distinct growth hormone binding proteins, the 22 kDa hGH variant represents the predominant isoform (Baumann 1999). Additionally, the sequence of the 22 kDa hGH isoform (AA 1 – 191 ; Figure 1) is proteolytically processed resulting in different mole- cular weight fragments. Some of these fragments were found to show specific biological properties (Salem 1988; Frigeri et al. 1988; Salem and Wolff 1989; Lewis et al. 1991; Hettiarachchi et al. 1997), a fact that suggested a pro-hormone role for the 22kDa isoform (Sinha and Jacobsen 1994; Haro et al. 1996; ISSN 0897-7194 print/ISSN 1029-2292 online q 2009 Informa UK Ltd. DOI: 10.1080/08977190903110121 Correspondence: R. Gutie ´rrez-Gallego, Parque de Investigacio ´ n Biome ´dica de Barcelona, C/Doctor Aiguader 88, E-08003 Barcelona, Spain. Fax: 34 93 316 04 67. E-mail: rgutierrez@imim.es Growth Factors, October 2009; 27(5): 255–264 Growth Factors Downloaded from informahealthcare.com by Universitat Pompeu Fabra on 12/28/11 For personal use only.