A Novel and Highly Divergent Homolog of Human Eosinophil Granule Major Basic Protein

Abstract

Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C 4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.

Major Basic Protein Homolog (MBP2): A Specific Human Eosinophil Marker

“…Comparisons of these proteins' cationicity also reveal distinctions. The isoelectric point (pI) of human MBP2, pI ϭ 8.7, differs considerably from that of human MBP1, pI ϭ 11.4 (2). In contrast, the isoelectric points of guinea pig MBP1, pI ϭ 11.7, and guinea pig MBP2, pI ϭ 11.3 (6,7), are quite similar, as are the isoelectric points of murine MBP1, pI ϭ 10.5, and MBP2, pI ϭ 9.95 (8).…”

“…In contrast, the isoelectric points of guinea pig MBP1, pI ϭ 11.7, and guinea pig MBP2, pI ϭ 11.3 (6,7), are quite similar, as are the isoelectric points of murine MBP1, pI ϭ 10.5, and MBP2, pI ϭ 9.95 (8). Despite their different isoelectric points, the in vitro biological effects of human MBP1 and MBP2 appear similar, e.g., cell killing, inducing superoxide anion production, and IL-8 release from neutrophils, and inducing histamine and leukotriene C4 release from basophils, but human MBP1 appears to be more potent than MBP2 in these activities (2).…”

“…The secondary granules of human eosinophils contain several proteins, including the major basic protein (MBP1), 3 MBP homolog (here termed MBP2, previously denoted hMBPH), eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase (1,2). Individually and collectively, these proteins likely damage tissues in diseases, such as asthma and atopic dermatitis, and also damage large multicellular parasites, such as microfilaria (3)(4)(5).…”

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils

“…Basophil-containing mononuclear cell fractions were isolated by density gradient centrifugation from freshly drawn venous blood of healthy adult donors according to a protocol approved by the Institutional Review Board of Rush University Medical Center. Basophils were enriched by negative selection using immunomagnetic beads (Basophil Isolation Kit; Miltenyi Biotec, Auburn, CA) and the miniMacs magnetic cell separation system (Miltenyi Biotec) according to the manufacturer's instructions; cells enriched by this method are 59 -88% basophils (37). For some experiments basophils were further purified by countercurrent elutriation and two-step Percoll density gradient followed by negative selection (24), resulting in 98 -99% purity by Alcian blue staining.…”

Eosinophil Major Basic Protein-1 Does Not Contribute to Allergen-Induced Airway Pathologies in Mouse Models of Asthma

“…MBPs are small cationic proteins (ϳ12 kDa; pI 10 -12) that colocalize with the characteristic electron-dense crystalline core of eosinophil secondary granules (11). In the mammalian orders examined to date, including Primata (humans) and Rodentia (mice), MBPs are encoded by two genes that share considerable sequence identity (12)(13)(14). The relative function(s) of each MBP in a given species is not known.…”