Anders Lindmark

Biosynthesis, processing and sorting of neutrophil proteins: insight into neutrophil granule development

Abstract: Neutrophil granulocytes are specialized phagocytic cells that carry a collection of granules for regulated secretion, each with distinct constituents. The granules can be classified as azurophil (primary), developed first, followed in time by specific (secondary) granules gelatinase granules, and secretory vesicles. Stage‐ and tissue‐specific transcription factors govern the successive expression of genes for granule proteins to allow storage of the gene products in these organelle categories whose packaging is separated in time. Many of the granule proteins, in particular those of the heterogeneous lysosome‐like azurophil granules, are subject to extensive post‐translational proteolytic processing into mature proteins, most commonly as a post‐sorting event. A selective aggregation of proteins destined for storage in granules, as discussed in this review, would facilitate their retention and eliminate a need for distinct sorting motifs on each granule protein. Aggregation of granule proteins, that are often cationic, would be assisted by the anionic serglycin proteoglycans present in neutrophils. The antibacterial granule proteins can serve as models for antibiotics and some of them possess a potentially useful therapeutic ability to bind and neutralize endotoxin. Because aberrant expression of transcription factors regulating the synthesis of granule proteins is often found in leukemia, the clarification of mechanisms regulating the timed expression of granule proteins will shed light on the maturation block in myeloid leukemias.

Processing and targeting of granule proteins in human neutrophils

Neutrophils contain an assembly of granules destined for regulated secretion, each granule type with distinct constituents formed before terminal differentiation. The earliest granules are designated azurophil (primary), followed in time by specific (secondary), and gelatinase granules as well as secretory vesicles. Transcription factors regulate the genes for the granule proteins to ensure that expression of the gene products to be stored in different organelles is separated in time. Similar to lysosomal enzymes, many granule proteins, in particular those of the heterogeneous azurophil granules, are trimmed by proteolytic processing into mature proteins. Rodent myeloid cell lines have been utilized for research on the processing and targeting of human granule proteins after transfection of cDNA. Results from extensive work on the hematopoietic serine proteases of azurophil granules, employing in vitro mutagenesis, indicate that both an immature and a mature conformation are compatible with targeting for storage in granules. On the other hand, the amino-terminal propeptide of myeloperoxidase facilitates both the export from the endoplasmic reticulum and targeting for storage in granules. Similarly, targeting of defensins rely on an intact propeptide. The proteolytic processing into mature granule protein is most commonly a post-sorting event. Mis-sorting of specific granule proteins into azurophil or lysosome-like granules can result in premature activation and degradation, but represents a potential for manipulating the composition and function of neutrophil granules.

Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines.

Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti‐neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegeners granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre‐proproteinase 3 cloned from U‐937 cells was transfected to the rat basophilic/mast cell line RBL‐1 and the murine myeloblast‐like cell line 32D cl3. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32‐kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N‐glycosidase F showed that both potential N‐glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino‐terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA‐containing sera from patients with Wegeners granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29‐kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA. J. Leukoc. Biol. 61: 113–123; 1997.

CHARACTERIZATION OF THE BIOSYNTHESIS, PROCESSING, AND SORTING OF HUMAN HBP/CAP37/AZUROCIDIN

Azurocidin is a multifunctional endotoxin‐ binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL‐1 and the murine myeloblast‐like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5‐kDa form disappeared, while the 37‐kDa form was further processed proteolytically, as judged by digestion with N‐glycosidase F. Conversion of high‐mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino‐terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly‐Phe‐diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino‐ terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino‐terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino‐terminal processing of human azurocidin. J. Leukoc. Biol. 66: 634–643; 1999.

Processing and intracellular transport of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937-modulation by brefeldin A, ammonium chloride, and monensin.

The effects of brefeldin A, monensin, and the weak base NH4C1 on the biosynthesis and processing of cathepsin G and neutrophil elastase of myeloid cells were investigated. Monoblast‐like U‐937 cells were biosynthetically labeled with [35S]methionine, followed by subcellular fractionation, immunoprecipitation, and analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophore‐sis and fluorography. Brefeldin A inhibited proteolytic processing, intracellular transport, and secretion. The effects were reversible inasmuch as removal of brefeldin A resulted in a normal pattern of processing and transfer to high‐density organelles, corresponding to lysosomes, and restitution of constitutive secretion of precursor forms. Both cathepsin G and neutrophil elastase acquired resistance to endoglycosidase‐H, suggesting that conversion to complex oligosaccharide side chains also occurs in the presence of brefeldin A. Monensin and NH4Cl inhibited final proteolytic processing, indicating either that acidification is necessary for directing cathepsin G and neutrophil elastase to lysosomal‐like organelles or that the protease(s) responsible for processing requires an acid pH. We conclude that pH‐dependent proteolytic processing of cathepsin G and neutrophil elastase occurs in post‐Golgi structures and that transfer to lysosomes or an immediately prelysosomal compartment is mandatory for complete processing. J. Leukoc. Biol. 55: 50–57; 1994.

Identification of a novel and myeloid specific role of the leukemia-associated fusion protein DEK-NUP214 leading to increased protein synthesis.

The t(6;9)(p22;q34) chromosomal translocation is found in a subset of patients with acute myeloid leukemia (AML). The translocation results in a fusion between the nuclear phosphoprotein DEK and the nucleoporin NUP214 (previously CAN). The mechanism by which the fusion protein DEK‐NUP214 contributes to leukemia development has not been identified, and disruptions of normal cellular functions by DEK‐NUP214 have previously not been described. In the present study, a novel effect of the DEK‐NUP214 fusion protein is demonstrated. Our findings reveal a substantial increase in global protein synthesis in DEK‐NUP214 expressing cells. Furthermore, we conclude that this effect is not the result of dysregulated transcription but merely due to increased translation. Consistent with the association with AML, the increased protein synthesis mediated by DEK‐NUP214 is restricted to cells of the myeloid lineage. Analysis of potential mechanisms for regulating protein synthesis shows that expression of DEK‐NUP214 correlates to the phosphorylation of the translation initiation protein, EIF4E. The present data provide evidence that increase of translational activity constitutes a mechanism by which the leukemogenic effect of DEK‐NUP124 may be mediated.

Rapid and effective CD3 T-cell depletion with a magnetic cell sorting program to produce peripheral blood progenitor cell products for haploidentical transplantation in children and adults.

BACKGROUND: Effective T‐cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting–based direct large‐scale T‐cell depletion, which is an attractive alternative to standard PBPC enrichment procedures.

A proposed plan for the management of adult sibling donors.

Siblings who are asked to donate haematopoietic stem cells are in a vulnerable position. Potential sibling donors must be fully informed, orally and in writing, and be able to provide informed consent without pressure or coercion.1, 2, 3 To achieve this, one must focus on the management of confidentiality in terms of donor volunteer status and strive to protect the integrity of the sibling.4, 5 At our centre we decided to improve this aspect in the care of potential sibling donors, enabling an early termination of the process for those not willing or unable to donate. We also wanted to improve the information to, and the follow-up of sibling donors.

Autologous del(20q)-positive erythroid progenitor cells, re-emerging after DLI treatment of an MDS patient relapsing after allo-SCT, can provide a normal peripheral red blood cell count

Summary:A 54-year-old RhD-negative male with del(20q)-positive myelodysplastic syndrome was transplanted with bone marrow from an HLA-identical RhD-positive sibling donor. Cytogenetic relapse was detected 21 months after stem cell transplantation (SCT), with reappearance of the original del(20q)-positive clone and reversion to recipient RhD-negative blood group. The patient received sequential donor lymphocyte infusions (DLIs), resulting in mild graft-versus-host disease and pure red cell aplasia. At 2 years post DLI, the patient remains in a stable condition, despite a dominance of recipient-derived erythro- and granulopoiesis originating in del(20q)-carrying progenitor cells. We conclude that reappearance of autologous erythropoiesis, upon relapse after allogeneic SCT, may be predictive of erythropenia after DLI and that re-emerging autologous del(20q)-positive erythropoiesis post DLI can provide a normal peripheral red blood cell count. Furthermore, in patients relapsing after blood-group-mismatched transplantation, a possible reversion to recipient blood group should be considered prior to blood transfusion or DLI.

Effective CD3 T-cell depletion using the CliniMacs (R) System to produce peripheral blood progenitor cell products for haploidentical transplantation in 23 children and adults: the updated Lund experience

Hematopoietic Cell Transplantation Comorbitdity Index (HCT-CI), is a score validated by Sorror et al. to evaluate transplant Transplant-Related Mortality (TRM) incidence, in patients undergoing Allogeneic Transplant. The aim of our study was to assess TRM and the reliability of this score in AML elderly patients undergoing Autologous Transplant. We have treated 101 elderly AML patients aged over 60 years, diagnosed between june 1999 and September 2009. All patients received an induction and consolidation treatment including HD-ARA-C and Idarubicin, with a 30% reduction dose in over 70 years patients; those obtaining a successful mobilization (CD34 + cell > 3 × 106/kg) underwent Autologous Transplant, 12 after Busulfan plus Melfalan (BUMEL) and 9 after BEAM conditioning. The remaining patients received alternative consolidation treatment. We obtained a 71% CR, 49 patients were in CCR and eligible to a second consolidation; 21 of these (43%) obtained a successful CD34 mobilization and underwent Autologous Transplant. Six patients had a HCT-CI 0 score, 11 had a score between 1 and 2, 4 patients had a > 3 score. One of the latter patients had a previous cancer, 2 had an heart valve disease and 1 had diabetes, peptic ulcer requiring treatments and previous severe infectious complications. Five out 21 patients died of transplant, with a 25% cumulative 1-year TRM incidence. Surprisingly no patients with a > 3 HCT-CI score died, vs. one patient in the 1–2 score group (9%) and 4 patients in the 0 score group (67%) (P = 0.02). Age < and > 70 years did not affect the TRM rate, but all the fi ve deaths occurred after the administration of Busulfan plus Melfalan (BUMEL) with an higher TRM rate in this group (42%, P = 0.04) in comparison with the patients receiving BEAM. In our small series all 6 patients with a 0 HCT-CI score received BUMEL as conditioning (P = 0.01), suggesting that the higher TRM rate in this group might be related to the administration of a more toxic conditioning regimen. Elderly patients generally have several comorbidities and HCTCI score might be an insuffi cient tool in assessing their frailty. The high TRM rate in elderly AML patients suggests that some of these patients should receive less toxic conditioning or alternative consolidation regimen. Larger retrospective and multicentric studies are therefore needed in order to better validate this score in this setting.