Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes

Abstract

The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system.

Figure 1. Sequence relationships of class-2 SPATE proteins.

The phylogenetic analysis of the aminoacidic sequence of class-2 SPATEs shown here was modified from reference , with permission of the Editorial board. The phylogenetic tree shows allelic variants of SPATEs in clusters (colored branches) Bacterial species and pathotypes from which SPATE sequences were derived are shown on the right side. Class-2 SPATEs also include a cluster of proteases found mostly in animal pathogens, which lack of the classic domain-2 (green branches, see text). SPATE members of each major cluster purified in this study are indicated with asterisks.

Figure 2. Class-2 SPATEs trigger leukoagglutination.

1×10⁵ Jurkat T cell were incubated with 2 µM of each class-2 SPATE at 37°C in a CO₂ incubator. One hour following initial treatment, cells were analyzed in an inverted light microscopy. Micrographs were taken in the light field at 20× magnification.

Figure 3. Binding of class-2 SPATEs to leukocyte subpopulations.

A, Multicolor flow cytometry was used to analyze direct binding of class-2 SPATE members (Pic, the protease deficient PicS258A, Tsh/Hbp and SepA) to different leukocyte subpopulations. Leukocytes were incubated with 2 µM of each FITC-labeled SPATE for 30 minutes on ice. To differentiate for specific leukocyte subpopulations, monocytes, T lymphocytes, and B lymphocytes were simultaneously stained with dye-conjugated antibodies directed against CD14, CD3 and CD19, respectively. Granulocytes were only selected by gating. B, Inhibition of SPATE binding by heat treatment and by competitive assays. Binding of FITC-labeled PicS258A, Tsh and SepA were tested as above (shaded in blue) and in the following conditions: as FITC-denatured SPATEs (outlined in red), or in a competitive binding with equimolar concentrations (2 µM) of unlabeled versus FITC-labeled SPATEs (shaded in orange). Untreated leukocyte populations are shaded in black. Histograms of FITC-SPATE binding on leukocytes are shown. Flow cytometry data are representative of at least three independent experiments.

Figure 4. Broad spectrum activity of class-2 SPATEs on mucin-type O-glycoproteins of the immune system.

3 µg of recombinant glycosylated proteins from human origin were incubated with 2 µM of each purified SPATE at 37°C for 1 h. Samples were analyzed by 4–20% gradient SDS/PAGE and blue Commassie staining. SPATEs and intact glycoproteins are indicated with arrows. Tsh/Hbp was also tested at 4 µM indicated with (h).

Figure 5. Class-2 SPATEs do not degrade non mucin-type, O-linked glycoproteins.

A, In order to assess the proteolytic activity of class-2 SPATEs on other heavily glycosylated proteins, we treated CD97, CD99, E-selectin, αβ-integrin and fibronectin with 2 uM class-2 SPATEs by overnight incubation at 37°C. Samples were analyzed by SDS-PAGE and Blue Commassie staining. B, Proteolytic activity of SPATEs was also evaluated on leukocyte markers routinely used in flow cytometry assays. 1×10⁶ total leukocytes were treated with 2 uM of SPATEs for 1 h at 37°C and analyzed by multi-color flow cytometry. The data show cleavage profiles against CD16(used in NK gating), CD14(monocytes), CD3(T lymphocytes), CD8(Cytotoxic lymphocytes) and CD19(B lymphocytes), and are representative of more than 5 independent experiments.

Figure 6. Class-2 SPATEs degrades the extracellular domain of mucin-type O-glycoproteins on human leukocytes subpopulation.

1×10⁶ total leukocytes isolated from human blood were incubated with 2 µM of purified SPATEs at 37°C for 1 h. Multi-color flow cytometry was used to analyze cleavage of glycopoteins in different leukocyte subpopulations. Cell types were identified through “forward and side scatter” gates plus cell type-specific lineage markers. Monocytes, T, and B lymphocytes were concurrently stained with conjugated antibodies directed against CD14, CD3 and CD19, respectively. Granulocytes were selected by gating. NK cells were first negatively selected for binding of anti-CD3 and then positively selected for binding of anti–CD16 and anti–CD56. The data show cleavage profiles of CD43, CD44, CD45 and CD162, and are representative of 3 independent experiments.

Figure 7. Cleavage of CD43 and CD162 glycoproteins in Jurkat T cells by class-2 SPATEs.

To visualize degradation of mucin-type glycoproteins by SPATEs, Jurkat T cells were treated with 2 µM of each SPATE for 1 h at 37°C. T cells were stained using fluorescent monoclonal antibodies against human CD43 (green) and CD162 (red) simultaneously, and analyzed by fluorescence microscopy. CD43 shows up in green, CD162 in red and co-localization of both, CD43 and CD162 is in yellow. DAPI was used to visualize cell nucleus (blue). Micrographs were taken at 40× magnification.

Figure 8. Reduced proteolytic activity of SPATE on deglycosylated proteins.

A, Upper panel; purified CD44, CD162 and C1-Inh glycoproteins were deglycosylated with deglycosylation enzyme cocktail by overnight at 37°C under denaturing (D) and non-denaturing (N) conditions. Extent of deglycosylation was assessed by comparison with untreated glycoproteins and by mobility shifts on SDS-PAGE gels. Lower panel; 1 µg of non-denaturing deglycosylated proteins were treated with 2 uM of each class-2 SPATE by 2 h at 37°C and analyzed by SDS-PAGE and blue Commassie staining. B, The overall sialic acid carbohydrate depletion on human leukocytes following treatment with 2 µM class-2 SPATEs during 1 h at 37°C was assessed by flow cytometry. Data show the percentage of sialic acid-bearing granulocytes and lymphocyte populations, and are representative of three independent experiments.

Figure 9. Conserved residues in the region spanning the catalytic triad of class-2 SPATEs are important for glycoprotease activity.

A, The aminoacid sequence of the region spanning the catalytic triad in domain-1 of class-2 SPATEs, were aligned with Clustal-omega. The residues comprising the catalytic triad are shaded in black (His), cyan (D) and red (S). The conserved catalytic motif of SPATEs are shaded in grey. Aminoacids shaded in yellow (conserved in most class-2) and blue (non conserved in all SPATEs) were mutated to those occurring in SepA by site directed mutagenesis. B, Positions of mutated residues (spheres) with respect to the catalytic triad (little spheres and sticks) in Pic, are depicted in the stereo ribbon structure of Hbp (PDB, WXR) . In orange, the domain-2 characteristic in most class-2 SPATEs, but not in AdcA is shown. C, Secretion of Pic derivatives harboring single site mutations and their activity on glycoproteins. Concentrated supernatant proteins from HB101 expressing Pic species were analyzed by SDS-PAGE and Commassie staining (top gel). 3 ug of glycoproteins (BSM, CD162, and CD45) were incubated with 10 µL of each supernatant for 2 h at 37°C, and analyzed as above (bottom gels). BSM, bovine submaxillary mucin.