Fig 1.
Protein alignment and illustration of functional domains and amino acids.
(A) Alignment of the secretory mDNase1 family members by Clustal Omega: the multiple sequence alignment tool of EMBL-EBI. The numbering of essential amino acids refers to the mature mDNase1 protein, most of which were discovered for bovine and human DNase1. The length of the pre-mature and mature sequence is indicated in brackets. References: DNase1 disulfide bridges and Ca2+-binding [8, 9], DNase1 N-glycosylation [10], DNase1 DNA binding [11, 12], DNase1 catalytic center and Mg2+ binding [9, 11, 13, 14], DNase1 actin binding [15], DNase1L3 nuclear localization signals [16], (B) Wild-type S. cerevisiae α-mating factor signal prepro-peptide (αMF-SP) in comparison to that in pPinkα-HC of the PichiaPinkTM pastoris expression system [17].
Table 1.
Primers.
Fig 2.
Establishment of rmDNase1L2 expression.
(A) Detection of a single transgene integration for αMF-DNase1L2 (αMF-D1L2) in three transformed clones by expression cassette specific PCR (αMF-D1L2: 1197 bp, pos. Ctrl: vector, neg. Ctrl: water, marker: GeneRuler 1 kb DNA Ladder). (B) Detection of mature rmDNase1L2 (D1L2, ~34 kDa vs calculated 29 kDa) in the SN of MeOH-induced clones by SDS-PAGE and Coomassie staining. Note the heat-dependent instability of rmDNase1L2 in Laemmli sample buffer [60] as shown in (C). (D) Enhanced expression level of mature rmDNase1L2 by doubling the expression cassette for αMF-DNase1L2 in the transformed vector (1x vs 2x). (E) Functionality of rmDNase1L2 as verified by DPZ. Note the high MM nuclease smear with its lowest band at ~48 kDa representing pre-mature rmDNase1L2 that is still fused to the hyper-tri-N-mannosylated pro-peptide of αMF-SP (αMF-D1L2). (F) De-N-glycosylation (ΔN) by PNgaseF or EndoHf confers the high MM nuclease smear to a single ΔN-αMF-DNase1L2 nuclease band (~41 kDa) in DPZ (ΔN-αMF-D1L2: pre-mature rmDNase1L2 of ~34 kDa in fusion to the de-N-glycosylated 7 kDa pro-peptide of αMF-SP, Fig 1A and 1B). Marker: PageRuler™ Prestained Protein Ladder in (B, D, E, and F) and Cozy Prestained Protein Ladder in (C).
Table 2.
Biochemical features of the secretory murine DNase1 family members.
Fig 3.
Co-expression of αMF-DNase1L2 with KEX2 or soluble KEX2-ΔTM613.
(A) Detection of the different transgene integrations in transformed clones by expression cassette specific PCR (αMF-D1L2: 1197 bp, KEX2: 2597 bp, KEX2-ΔTM613: 2034 bp, pos.Ctrl: vector, neg.Ctrl: water, marker: GeneRuler 1 kb DNA Ladder). (B) Detection of rmDNase1L2 (D1L2) in the SN of MeOH-induced clones by SDS-PAGE and Coomassie staining reveals rmDNase1L2 degradation in clones co-expressing Golgi-located full-length KEX2. (C) Degradation of rmDNase11L2 also occurs in the presence of soluble truncated KEX2-ΔTM613. Marker in (B, C): PageRuler™ Prestained Protein Ladder.
Fig 4.
Optimization of rmDNase1L2 expression.
(A) Washing cells with PBS, pH 6.0, between growth and expression (MeOH containing SCM-Ade medium) increased the expression of rmDNase1L2 (D1L2) independently of the growth medium (BMGY or SCG-Ade). (B) Dependence of rmDNase1L2 expression on the concentration of Kaiser`s synthetic complete adenine drop-out mixture in the SCM-Ade expression medium. SCG-Ade growth medium was used. (C) Replacement of SCM-Ade by peptone methanol (PM) expression medium enhances the expression of rmDNase1L2 at an optimal concentration of 0.5% (w/v) peptone. (D) Dependence of the expression level and processing of pre-mature αMF-DNase1L2 (αMF-D1L2) to mature rmDNase1L2 on the nutrient content of the growth medium. Compared are 1x SCG-Ade (0.2% (w/v) Kaiser’s SC-Ade) and BMGY medium (1x: 1% (w/v) yeast extract, 2% (w/v) peptone, YP) with varying YP-concentrations. (E, F) Expression of rmDNase1L2 using 1x SCG-Ade growth and PM-expression medium with 0.5% (w/v) peptone. (E) Stability of mature rmDNase1L2 depends on the pH-value of the expression medium (0.1 M potassium phosphate buffer). (F) Influence of the temperature and duration of the expression culture on the expression level of mature rmDNase1L2. In all experiments, the basal expression pattern of P. pastoris transgenic for mDnase1l2 without MeOH induction was evaluated in parallel (Ctrl). Marker in (A-F): PageRuler™ Prestained Protein Ladder.
Fig 5.
Purification of rmDNase1L2 from culture supernatant.
(A) DEAE-cellulose anion-exchange chromatography of filtrated and diluted SN. (FT: flow through, E50-500: elution fraction at 50–500 mM NaCl). (B) Purification of mature rmDNase1L2 (rmD1L2, D1L2) from DEAE elution fraction E200 by Heparin-Sepharose affinity chromatography. Final purification of mature rmDNase1L2 from E1500 was done by gel-filtration (last lane). (A, B) Top image: Coomassie-stained SDS-gel, lower image: absolute activity of the samples measured by HCA. (C) Successful separation of mature rmDNase1L2 from pre-mature αMF-DNase1L2 (αMF-D1L2) as shown by DPZ. The final pure rmDNase1L2 protein co-migrates with its native form (nmD1L2) present in a cutaneous tissue homogenate of a Dnase1/Dnase1l3 double KO mouse (20 μg protein loaded). Marker in (A-C): PageRuler™ Prestained Protein Ladder.
Fig 6.
Establishment of expression and purification of rmDNase1 from culture supernatant.
(A) Expression and processing of pre-mature αMF-DNase1 (αMF-D1, ~51 kDa) to mature rmDNase1 (D1, ~37 kDa) depends on the choice and nutrient content (1x BMGY: 1% yeast extract, 2% (w/v) peptone) of the growth medium (PM expression medium was used). (B) Temperature, time and protease dependent processing of pre-mature αMF-DNase1 to mature rmDNase1 in dialyzed SN in the presence and absence of the protease inhibitor AEBSF. (C) Purification of mature rmDNase1 from dialyzed and processed SN by DEAE-cellulose anion-exchange chromatography (FT: flow through, E50-500: elution fraction at 50–500 mM NaCl). (A-C) Top image: Coomassie-stained SDS-gel, lower image: specific activity of the samples measured by HCA. (D) Successful separation of mature rmDNase1 from pre-mature αMF-DNase1 as shown by DPZ. Purified rmDNase1 co-migrates with native mDNase1 (nmD1) present in a lacrimal tissue homogenate of a WT mouse (50 ng protein loaded). (E) Analysis of de-N-glycosylation by EndoHf and PNGaseF reveals di-N-glycosylation (mannosylation) of rmDNase1 by DPZ. (rmDNase1: di-N-glycosylated ~ 37 kDa, mono-N-glycosylated ~35 kDa and Δ-N-glycosylated ~ 33 kDa). Marker: PageRuler™ Prestained Protein Ladder.
Fig 7.
Establishment of expression and purification of rmDNase1L3 from culture supernatant.
(A) Expression and processing of pre-mature αMF-DNase1L3 (αMF-D1L3, ~49 kDa) to mature rmDNase1L3 (D1L3, ~35 kDa) is less dependent on the choice and nutrient content of the growth medium (1x BMGY: 1% yeast extract, 2% (w/v) peptone) than that of rmDNase1 and rmDNase1L2 (PM expression medium was used). (B) Purification of mature rmDNase1L3 from diluted SN by DEAE-cellulose anion-exchange and Heparin-Sepharose affinity chromatography (FT: flow through, E50-1500: elution fraction at 50–1500 mM NaCl). (A, B) Top image: Coomassie-stained SDS-gel, lower image: specific nuclease activity of the samples measured by HCA. (C) Successful separation of pre-mature αMF-DNase1L3 from mature rmDNase1L3 as shown by DPZ. The final pure rmDNase1L3 protein co-migrates with native mDNase1L3 (nmD1L3) present in splenic tissue homogenate of a Dnase1 KO mouse (40 μg protein loaded). (D) Direct comparison of the purified recombinant secretory DNase1 proteins by SDS-PAGE and Coomassie-staining (each 4 μg). (E) Detection of glycosylation by Periodic acid-Schiff staining of an SDS-PAGE gel. Only rmDNase1 (rmD1) is glycosylated whereas de-N-glycosylated rmDNase1 (ΔN-rmD1), rmDNase1L2 (rmD1L2) and rmDNase1L3 (rmD1L3) lack glycosylation. Left image: stained gel prior to washing, right image: stained gel after washing with diluted acetic acid. Marker: PageRuler™ Prestained Protein Ladder.
Fig 8.
Specific activities of the rmDNases.
Hyperchromicity assay at different pH-values in the presence of 0.1 mM CaCl2 and either 1 mM MgCl2 (A), CoCl2 (B) or MnCl2 (C). Equimolar amounts of the recombinant nucleases were tested (30 μM).
Fig 9.
Substrate specific activities of the rmDNases.
(A, B) Lambda DNA digestion at different pH-values in the presence of 2 mM CaCl2 and either 2 mM MgCl2 or MnCl2. Aliquots of the assay were analyzed by agarose gel electrophoresis and subsequent EtBr-staining. Equimolar amounts of the recombinant nucleases were tested (0.3 μM). Optimal pH-ranges are framed in red and the optimal pH-value estimated by HCA (Fig 8) is marked by an asterisk *. Consistent with the HCA, all three nucleases exert the same activity at pH 7.0 in the presence of Mn2+ ions, whereas at pH 7.5 in the presence of Mg2+ ions rmDNase1 (D1) exerts an activity, which is ten times higher as quantified by HCA. (B) In contrast to rmDNase1, heparin inhibits rmDNase1L2 (D1L2) and rmDNase1L3 (D1L3) in the protein-free lambda DNA assay at pH 7.0 in the presence of 2 mM CaCl2 and MnCl2. (C) In contrast to protein-free lambda DNA in (A), all three nucleases exert an almost equal ability to degrade chromatin at pH 7.5 in the presence of 2 mM CaCl2 and MgCl2 using cell nuclei of murine NIH-3T3 fibroblasts. However, the cleavage mode differs (rmDNase1: random, rmDNase1L2: bi-modular and rmDNase1L3: internucleosomal) as evaluated by agarose gel electrophoresis and subsequent EtBr-staining. Heparin accelerates the activity of rmDNase1 and inhibits that of rmDNase1L3 as previously shown [37]. In contrast to the lambda DNA assay, rmDNase1L2 is not inhibited by heparin but its cleavage mode changes from bi-modular to exclusively random.
Fig 10.
Interaction of monomeric α-actin with rhDNase1 and rmDNase1.
(A) Polymerization of monomeric skeletal muscle α-actin supplemented with fluorescent pyrenyl-labeled monomeric α-actin to microfilaments in the absence and presence of rhDNase1 (rhD1, Pulmozyme™, Roche) or rmDNase1 (rmD1) at equimolar ratio (2.5 μM). (B) Effect of increasing monomeric α-actin concentrations on the DNase1 activity as determined by HCA at pH 7.0 in the presence of 0.1 mM CaCl2 and 1 mM MgCl2.