Biochemical Aspects

In the late 1980s, after screening the human pancreatic complementary DNA (cDNA) library, Shak, et al. [12] found the cDNA encoding human DNase I. This cDNA was subsequently sequenced and expressed in human embryonic kidney 293 cells (HEK293), yielding rhDNase I, also called dornase alfa. The recombinant enzyme contains, as does purified human pancreatic DNase I, 260 amino acids, shows a 77% homology with bovine DNase I, and contains at its N terminus a 22-amino-acid-long sequence encoding a secretion signal [12,15] (Figure 5.2). The rhDNase I purified from the supernatant of transfected cells is a mixture of various glycosylated species with a pI between 3 and 4 and an average molecular mass of approximately 35 kDa [16]. Like bovine DNase I, rhDNase I contains four cysteine residues (amino acids 101, 104, 173, and 209) and two potential N-linked glycosylation sites, N18 and N106 [15,17,18]. Depending on the expressing cell type, one or both sites become glycosylated [19]. The carbohydrate chains, which are not essential for enzymatic activity, contain N-acetylglucosamine, sialic acid, mannose, fucose, and mannose-6-phosphate [20]. Similar to its bovine counterpart, rhDNase I contains two disulfide bridges [21]. The first one (C101-C104) is not essential for enzyme activity, while the second one (C173-C209) is essential for the structure and activity of the enzyme [17]. Close to this essential disulfide bridge, a strong binding site for Ca2+ is present in the loop formed by residues 201 to 207. Binding of Ca2+ to this site is mediated by the negatively charged side chain of D201, by the hydroxyl group of T203, and by the amide oxygens of T203, T205, and T207. A second strong binding site for Ca2+ is located near the flexible loop region (residues 100 to 105) that contains the nonessential disulphide bridge. The negatively charged side chains of D99, D107, E112, and the amide oxygen of F109 form this binding site. Mg2+ and Mn2+ can compete with Ca2+ for binding to one of these strong binding sites [22]. The affinity of these binding sites for Ca2+ decreases with pH; at pH 5.5, DNase I binds only one Ca2+ ion with high affinity. Upon heating, rhDNase I (10 mg/mL,

human MR GMKLLGALLA LAALLQGAVS LKIAAFNIQT FGETKMSNAT LVSYIVQILS bovine MR GTRLMGLLLA LAGLLQLGLS LKIAAFNIRT FGETKMSNAT LASYIVRIVR

40 50 60 70 80

human RYDIALVQEV RDSHLTAVGK LLDNLNQDAP DTYHYVVSEP LGRNSYKERY bovine RYDIVLIQEV RDSHLVAVGK LLDYLNQDDP NTYHYVVSEP LGRNSYKERY

90 100 110 120 130

human LFVYRPDQVS AVDSYYYD|DG CEPCGNDTFN REpAIVRFFS RFTEVREFAI bovine LFLFRPNKVS VLDTYQYDDG CESCGNDSFS REPAVVKFSS HSTKVKEFAI

140 150 160 170 180

human VPLHAAPGDA VAEIDALYDV YLDVQEKWGL EDVMLMGDFN AGCSYVRPSQ bovine VALHSAPSDA VAEINSLYDV YLDVQQKWHL NDVMLMGDFN ADCSYVTSSQ

190 200 210 220 230

human WSSIRLWTSP TFQWLIPDSA |DTTATPT|HCA YDRIVVAGML LRGAVVPDSA bovine WSSIRLRTSS TFQWLIPDSA DTTATSTNCA YDRIVVAGSL LQSSVVPGSA

240 250 260

human LPFNFQAAYG LSDQLAQAIS DHYPVEVMLK bovine APFDFQAAYG LSNEMALAIS DHYPVEVTLT

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