From the Departments of Immunology, Internal
Medicine, and Biochemistry and Molecular Biology, Mayo Clinic and Mayo
Foundation, Rochester, Minnesota 55905,
Incyte Pharmaceuticals,
Palo Alto, California 94304, and the ¶ Department of
Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center,
Chicago, Illinois 60612-3864
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ABSTRACT |
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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 C4 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.
The eosinophil functions as a major effector cell in pathologic
states (1, 2) including those associated with allergy and with
pronounced eosinophilia and as a protective cell against helminthic
infection (1). The major basic protein
(MBP)1 is an abundant granule
protein and an important mediator of eosinophil biological function
(1-3). It constitutes the crystalloid core of the eosinophil-specific
granule and is the principal protein composing the granule (3, 4).
Human MBP (hMBP) is a 13.8-kDa protein with a calculated isoelectric
point near pH 11. The cDNA sequence predicts initial translation of
a 25.2-kDa prepro-form containing an additional signal sequence
("pre") and a highly acidic ("pro") domain. MBP possesses
numerous cytotoxic properties (3) including the ability to damage
helminths, bacteria, protozoa, and mammalian cells, and it is
implicated as a mediator of pathology. For example, MBP is deposited on
damaged lung epithelium of patients with asthma, in the upper dermis of
the skin in patients with atopic dermatitis, and in cardiac tissues of
patients with eosinophilic endomyocardial disease (5-7). Furthermore,
MBP stimulates bronchial hyperreactivity similar to that observed in
asthma (3, 8, 9). MBP also activates cells and stimulates cytokine
production by them. Examples include the regulation of cytokine
production from neutrophils,2
eosinophils (11), and lung fibroblasts (12) and stimulation of
biological mediator release from basophils and mast cells (13). Additionally, blood concentrations of the pro-form of MBP (pro-MBP) increase dramatically during human pregnancy (14, 15), and this results
from a striking expression of pro-MBP by the placental X cell (16, 17).
Altogether, these observations support MBP's involvement in the
pathophysiology of allergic and eosinophil-associated diseases and its
beneficial role in resistance to parasitic disease.
To further characterize gene products important in eosinophil function,
we sought to identify novel genes transcribed by precursor blood cells
cultured in the presence of interleukin (IL)-5, a cytokine that causes
eosinophil proliferation, differentiation, and activation (3). The
approach chosen involved large scale cDNA sequencing (18) of
thousands of plasmid-inserted cDNAs generated from the
IL-5-differentiated precursor cells. The resulting frequency profile of
the sequenced cDNAs, referred to as a Transcript Image, provides a
quantitative and qualitative record of gene expression (18, 19).
Analysis of the Transcript Image of the IL-5-differentiated precursor
cells revealed a transcript with significant homology to human
prepro-major basic protein (hprepro-MBP). This transcript was named
hprepro-MBP homolog (hprepro-MBPH). Compared with previous MBP
molecules, the deduced amino acid sequence corresponding to the MBP
section of the hprepro-MBPH transcript showed a marked reduction in the
number of positively charged amino acids. This marked basicity
difference, the transcript's high abundance, hMBP's association with
various health and disease processes, and the possible contribution of
hMBPH to previously recognized hMBP activities prompted us to further
characterize this MBP-like molecule. We report here the identification,
isolation, and characterization of this novel, highly divergent homolog
of hMBP.
IL-5-induced Differentiation of Umbilical Cord Precursor
Cells--
Differentiating eosinophils were produced in
vitro by stimulation of umbilical cord precursor cells (UCC) with
recombinant human IL-5, a generous gift from Schering-Plow Research
Institute, Kenilworth, NJ, essentially as described before (20-23).
Eosinophil differentiation was monitored with cyanide-resistant
peroxidase staining (22), a marker for eosinophil peroxidase. By days
10-12, 60-100% of the cultured cells stained positive for eosinophil peroxidase. Cells from the recombinant human IL-5-stimulated cultures were pelleted and lysed in 4 M guanidine thiocyanate at
107 cells/ml. Lysates were stored at Transcript Image Generation--
Total RNA, prepared from
recombinant human IL-5-stimulated UCC populations following guanidinium
lysis as described above, was subjected to DNase treatment, and
polyadenylated RNA was isolated using Qiagen Oligotex (Qiagen,
Inc., Chatsworth, CA). Polyadenylated RNAs were converted to
cDNA using the SuperScript Plasmid System (Life Technologies,
Inc.), where the plasmid cloning vector was a polylinker-modified
pSPORT. Prior to cloning, cDNAs were size-fractionated on Sepharose
CL-4B (Amersham Pharmacia Biotech).
Following transformation, individual colonies were picked, and plasmid
DNA was released and purified using the REAL Prep 96 plasmid kit
(Qiagen). Sequencing of cDNA using ABI 377 sequencers and standard
ABI sequencing protocols was performed on 15,000 plasmid DNAs. All
plasmid sequences were screened to identify putative cDNA inserts
followed by iterative screening of these cDNAs against public
nucleic acid and protein data bases for sequence identification. This
process yielded a total of 8,146 useable sequences (i.e.
nonmitochondrial and nonribosomal sequences). The number of times a
unique cDNA was identified was recorded, resulting in a steady
state transcription profile or image, and referred to as a Transcript Image.
mRNA Expression in Tissues--
Polymerase chain reaction
(PCR) primers specific for the "pro" section of hprepro-MBP or
hprepro-MBPH were designed using Oligo software (National Biosciences,
Inc., Plymouth, MN). The hprepro-MBP primer pair
5'-TCTGAGACTTCCACCTTTGAGACC-3' and 5'-GTTTTTGTCCACCATATCTGGCAC-3' and
hprepro-MBPH primer pair 5'-AGAATGATGCCCCCCATCTG-3' and
5'-CACTGGAAGTCCTTGTCTAAGGCAG-3' were expected to produce PCR products
of 198 base pairs and 214 base pairs in length, respectively. Template
cDNAs from human tissues (CLONTECH, Palo Alto,
CA) were tested for the presence of hprepro-MBP or hprepro-MBPH
sequence: brain, bone marrow, colon, heart, kidney, lymph node, liver,
lung, ovary, peripheral blood leukocytes, pancreas, placenta, prostate,
skeletal muscle, small intestine, spleen, testis, thymus, and tonsil.
The Expand PCR System (Roche Molecular Biochemicals) was used with
cycling parameters of initial denaturation at 94 °C for 5 min, 10 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C
for 60 s followed by 28 cycles of 94 °C for 15 s, 57 °C
for 30 s, and 72 °C for 60 s with an additional 20 s
each cycle and a final extension time of 7 min at 72 °C. PCR
products were visualized with ethidium bromide in 2% agarose gels.
Electronic Northern--
The full-length cDNA sequence of
hprepro-MBPH was used to search for matching sequences (>95%
identity) in Incyte's assembled data base. This data base was composed
of Transcript Image data derived from multiple tissue samples divided
into 20 distinct tissue categories (see Table III). The total number of
sequence matches (i.e. "absolute abundance") and the
distribution of matches across the distinct tissue samples composing a
given tissue category (i.e. "found in") were recorded.
Monoclonal Antibody Production--
A peptide corresponding to a
segment of the N-terminal sequence of hMBPH (see below; Fig. 1) with
minimal homology to hMBP was synthesized and covalently coupled to
Imject maleimide-activated keyhole limpet hemocyanin (Pierce) in the
Mayo Protein Core Facility (Mayo Clinic, Rochester, MN). Keyhole limpet
hemocyanin peptide (approximately 50 µg) in RIBI adjuvant (RIBI
ImmunoChem Research, Hamilton, MT) was injected intraperitoneally into
five BALB/c mice (Charles River Laboratories, Wilmington, MA) monthly
for 3 months followed by a final intravenous injection of approximately 50 µg of keyhole limpet hemocyanin peptide in saline 3 days prior to
harvesting the spleens (25). Spleen cells were fused to FO myeloma
cells and cultured as described previously (25, 26). Conditioned media
were screened for reactivity to the immunizing peptide using the
F.A.S.T. system (Falcon Assay Screening Test; Becton Dickinson, Oxnard,
CA) with the immunizing peptide covalently coupled to
125I-labeled Imject maleimide-activated bovine serum
albumin (Pierce) (27). Antibodies were selected for specificity and
avidity using peptide alone immobilized on Immulon-4 Removawell
microtiter wells (Dynatech Laboratories, Chantilly, VA).
Hybridomas were subcloned using standard limiting dilution methods.
Verification of monoclonal antibody reactivity was further assessed by
slot blot analysis of immunizing peptide bound to Immobilon-P transfer
membrane (Millipore Corp., Bedford, MA). Ultimately, one hybridoma,
J191-12H11, was cultured, and the antibody was purified using a POROS
20 G - Protein G column (PerSeptive Biosystems, Framingham, MA).
Purification of hMBPH--
Eosinophils (about 1 × 1011 cells/patient, final purity of 90%) were collected by
cytapheresis of patients with the hypereosinophilic syndrome (28).
Cells were further processed to isolate eosinophil granules and granule
proteins, as described earlier (29-31). Fractions (1 ml each) from the
Sephadex G-50 column were probed for hMBPH and hMBP with monoclonal
antibodies J191-12H11 and J6-8A4, respectively, by Western blotting
essentially as described previously (26). Fractions containing hMBPH
were pooled and refractionated on Sephadex G-50. Fractions positive for
hMBPH with J191-12H11 and negative for hMBP with J6-8A4 by Western
blotting and showing a single band by Coomassie Brilliant Blue R-250
(Bio-Rad) staining were pooled, concentrated, and stored at N-terminal Amino Acid Sequencing--
The N-terminal amino acid
sequences of hMBPH and hMBP were analyzed by Edman degradation
microsequencing with the PE/Applied Biosystems 492 Procise Protein
Sequencing System (Perkin-Elmer Corp./Applied Biosystems Division)
using pulsed liquid chemistry in the Mayo Protein Core Facility. Data
were collected and analyzed with PE/Applied Biosystems model 610 Sequencing Software. The desalted protein samples were absorbed to
polyvinylidine difluoride membranes with the PE/Applied Biosystems
ProSorb cartridges before sequencing.
Mass Spectrometry--
A MAGIC 2000 HPLC system (Michrom
Bioresources Inc., Auburn, CA) fitted with a C8 column (50 × 0.5 mm; Michrom Bioresources Inc.) in series with a Finnigan MAT 900 double
sector mass spectrometer (Bremer, Germany) fitted with a position- and
time-resolved ion counter array detector was used to analyze purified
hMBPH and hMBP. A 15-min gradient from 5 to 60% mobile phase B (89.5%
acetonitrile, 7% water, 3% n-propanol, 0.5% acetic acid,
0.02% trifluoroacetic acid) in mobile phase A (2% acetonitrile,
97.5% water, 0.5% acetic acid, 0.018% trifluoroacetic acid) at a
flow rate of 30 µl/min was utilized, giving an estimated gradient
delay of 2 min. Effluent from the HPLC column was interfaced with the
MS via a fused silica capillary (180-µm outer diameter × 25 µm inner diameter; Polymicro Technologies, Phoenix, AZ) passed
coaxially through the inner bore of the electrospray needle. This
needle was biased at 4.8 kV with respect to the accelerating voltage of
5 kV. Ionization was by positive ion electrospray with N2
sheath gas flow to assist nebulization (1.5 liters/min). The mass
spectrometer was operated over a mass range of 500-2500 Da at a scan
rate of 5 s/decade. The position- and time-resolved ion counter
detector was operated at a microchannel voltage of 820 V and a mass
window of 8%.
For alkylation of protein prior to mass spectrometric analysis, hMBPH
and hMBP were incubated with iodoacetamide (IA; 20 mol of IA/mol of
protein) in 100 mM Tris, pH 8.0, at 37 °C for 30 min.
Similarly, reduction and alkylation were performed with dithiothreitol (20 mol/mol of protein) in 100 mM Tris, pH 8.0 at 37 °C
for 30 min followed by the addition of IA (40 mol/mol of protein) and continued incubation for 30 min.
Extinction Coefficient Determination--
Purified hMBPH or hMBP
in acetate buffer (25 mM sodium acetate, 150 mM
NaCl, pH 4.2) with an absorbance at 280 nm of about 0.75 was
centrifuged at 35,000 × g for 20 min just prior to the addition of the supernatant to a 1 × 1-cm quartz cuvette. An
absorbance spectrum (250-400 nm) was then acquired on a dual beam
Varian CARY 2200 spectrophotometer with acetate buffer as the blank. Three 50-µl samples were taken directly from the cuvette using three
separate calibrated pipettors. A portion of each sample was then
subjected to amino acid analysis in triplicate with postcolumn ninhydrin detection to quantitate the total mass of protein present. Briefly, the proteins were vapor-hydrolyzed with 6 M
hydrochloric acid and 1% phenol for 24 h under vacuum at
110 °C. The hydrolysates were analyzed on a Beckman 6300 amino acid
analyzer (Beckman Instruments) modified with the Pickering Laboratories
column, Na+ buffers (pH 3.28, 4.25, 6.40), and Trione
ninhydrin reagent (Pickering Laboratories, Mountain View, CA).
Norleucine was the internal standard, and a sample of standardized
bovine serum albumin (Pierce) was the hydrolysis control. Extinction
coefficients for hMBPH and hMBP were subsequently calculated from their
respective absorbance values at 280 nm corrected for light scattering
(32) (correction resulted in a 3-4% reduction in the uncorrected
absorbance values at 280 nm), and their average protein concentrations
were determined from the three 50-µl samples. The S.D. of the average
protein concentrations for hMBPH and hMBP were used to estimate the
errors of their respective calculated extinction coefficients.
Theoretical molar extinction coefficients ( Inhibition of Incorporation of
[14C]Leucine--
Cytotoxic effects of hMBPH and hMBP
were tested using human chronic myelogenous leukemia K562 cells
(CCL-243; American Type Culture Collection, Rockville, MD) essentially
as described earlier.3
Twenty-five microliters of these leucine-starved K562 cells (2.5 × 104 cells) were added in triplicate to a Costar
half-area, flat bottom 96-well plate (Costar Corp., Cambridge, MA)
followed by the addition of 2.5 µl of buffer or protein solution. The
plate was incubated at 37 °C and 5% CO2 for 2 h,
and then 0.1 µM [14C]leucine was added.
After a final incubation at 37 °C and 5% CO2 for 2 h, 100 µl of 0.1% SDS was added to each well; the contents were
mixed; and the solution was transferred to a 1.5-ml microcentrifuge tube. A wash of 100 µl of 0.1% SDS was added to each well, mixed, and transferred to its respective tube containing the first wash. Less
than 1% of the radioactive counts remained in the well. Bovine serum
albumin (100 µl of a 0.1% solution) was added to each tube followed
by 300 µl of 20% trichloroacetic acid to precipitate protein (34).
The tubes were incubated at 4 °C for 30 min and then centrifuged at
12,000 × g for 20 min. The resulting pellet was
dissolved in 100 µl of 1 M NaOH, 1 ml of Ultima Gold
scintillation fluid (Packard Instrument Co., Downers Grove, IL) was
added, and the samples were counted for
Inhibition of [14C]leucine incorporation was calculated
using the following equation: % inhibition of incorporation = (1 Neutrophil Superoxide Anion Production--
Neutrophils (2 × 105), isolated from venous blood of healthy adult donors
as described previously (35), were incubated with the indicated
concentrations of hMBPH or hMBP or with equivalent volumes of the
sodium acetate buffer, in HEPES-albumin-glucose buffer, pH 7.4, containing 0.6 mM CaCl2 and 1 mM
MgCl2 (HAGCM buffer) and 80 µM
ferricytochrome C (Sigma) for 30 min at 37 °C in an oscillating
water bath. Total reaction volume was 0.2 ml. The reactions were
stopped by centrifugation at 400 × g for 10 min at
4 °C, and superoxide anion (O Neutrophil IL-8 Production--
Neutrophils (106),
isolated as described above under sterile conditions, were incubated in
24-well tissue culture plates (Costar, Cambridge, MA) with the
indicated concentrations of hMBPH or hMBP, with equivalent volumes of
the sodium acetate buffer or 100 ng/ml lipopolysaccharide
(Escherichia coli 055:B5) (Sigma) in RPMI 1640 containing
100 µg/ml each of penicillin and streptomycin, 2 mM L-glutamine (Life Technologies), and 10% heat-inactivated
autologous serum for 20 h at 37 °C in a 5% CO2
atmosphere as described earlier.2 Total culture volume was
0.5 ml. Culture supernatants were collected by centrifugation at
400 × g for 10 min at 4 °C and were stored at
Basophil Histamine and Leukotriene C4
Release--
Basophil-containing mononuclear cell fractions were
isolated from venous blood of healthy adult donors as described
previously (36). Basophils were enriched by negative selection using
magnetic beads (Basophil Isolation Kit; Miltenyi Biotec, Auburn, CA)
and the midiMACS magnetic cell separation system (Miltenyi Biotec, Auburn, CA) as described by the manufacturer. The percentage of basophils in the three experiments ranged from 59 to 88% by Alcian blue staining. Basophils (7 × 104) were incubated
with the indicated concentrations of hMBP or hMBPH or with volumes of
vehicle buffer (25 mM sodium acetate, 0.15 M
NaCl, pH 4.2) equal to those required for protein addition in HAGCM
buffer for 30 min at 37 °C in an oscillating water bath. Total
incubation volume was 0.2 ml. The reactions were stopped by the
addition of 0.3 ml of cold HEPES-albumin-glucose buffer, pH 7.4, and
centrifugation at 1000 × g for 2 min at room
temperature. The leukotriene C4 (LTC4) content
in an aliquot of the cell-free supernatant was measured using an enzyme
immunoassay kit for LTC4/leukotriene D4/leukotriene E4 (Amersham Pharmacia Biotech)
as described previously (36), and results are expressed as ng of
immunoreactive LTC4/106 basophils. At the
basophil concentration used in this study, the LTC4
released by the basophils is not metabolized to leukotriene D4 or leukotriene E4 (37). Histamine content in
the remainder of the cell-free supernatant was determined
fluorometrically, and histamine release was calculated as the
percentage of total histamine content as described previously (36).
Spontaneous histamine and LTC4 release was determined with
cells incubated in the absence of stimulus.
Identification of Human prepro-MBP Homolog cDNA--
Table
I shows the 23 most abundant transcripts
present in IL-5-differentiated UCC as determined by Transcript Image
methodology. Three of the four most abundant transcripts were
essentially identical to eosinophil-specific mRNA sequences, namely
hprepro-MBP, Charcot-Leyden crystal protein, and eosinophil-derived
neurotoxin (EDN). Another eosinophil-specific sequence, eosinophil
peroxidase, was the eighth most abundant sequence. The Transcript Image
also showed the presence of a novel sequence with 49% homology at the
protein level to hprepro-MBP (Table I). This sequence was designated
hprepro-MBPH. The hprepro-MBP and hprepro-MBPH cDNA sequences were
the first and fifth most abundant cDNA inserts of the 8,146 clones
screened and comprised 8.1 and 1.1% of these cDNA inserts,
respectively (Table I). Fig. 1 shows the
cDNA and deduced amino acid sequence of hprepro-MBPH. The coding
region of the nucleotide sequence for hprepro-MBPH is 64% homologous
to that of hprepro-MBP when optimally aligned using the MacVector 4.5 program (Eastman Kodak Co.). Consistent with the cDNA structure of
hprepro-MBP, the homolog cDNA codes for an N-terminal signal
(i.e. "pre") sequence, a highly acidic "pro"
section, and a basic "MBP" section (Fig. 1 and Table II). In contrast, due to the diminished
basicity of its MBP section, the isoelectric points of hpro-MBPH and
hMBPH are approximately 100-fold lower than those of hpro-MBP and hMBP,
respectively (Table II).
Tissue mRNA Expression of hprepro-MBP and
hprepro-MBPH--
From the cDNA sequences for hprepro-MBP and
hprepro-MBPH, PCR primers specific for each pro section were designed
(see "Experimental Procedures" and Fig. 1). Fig.
2A shows the specificity of
the primer pairs. First-strand cDNAs derived from various tissue
types, normalized on the basis of housekeeping genes, were used as
templates in PCRs (see "Experimental Procedures"). PCR products
were subsequently analyzed electrophoretically. Fig. 2, B
and C, show representative results for a portion of the
tissues tested for mRNA expression. Amplification of a DNA fragment
of the expected length (198 nucleotides for hprepro-MBP and 214 nucleotides for hprepro-MBPH) at a cycle number of 38 was observed from
bone marrow and placenta (Fig. 2B) and bone marrow only
(Fig. 2C) for hprepro-MBP and hprepro-MBPH, respectively.
The tissue expression pattern for hprepro-MBP is consistent with
previous reports (17, 39). Notably, no expression was detected in
peripheral blood leukocytes, as expected, because of their terminal
differentiation (40).
As a second assessment of the hprepro-MBPH tissue expression profile,
an electronic Northern was performed using the homolog sequence as the
query sequence (see "Experimental Procedures" and Table
III). Consistent with the PCR data of
Fig. 2, expression of the homolog was only detected in the
IL-5-differentiated UCC transcript image data set within the hemic and
immune system tissue category. This analysis included an absence of
matches with Transcript Image data from multiple placenta samples
contained in the "embryonic structures" tissue category.
Purification and Identification of hMBPH from Eosinophil
Granules--
Using the deduced amino acid sequence of hpro-MBPH, an
antibody (J191-12H11) specific for an N-terminal sequence of hMBPH (see above; Fig. 1) was isolated and used to probe gel filtration column fractions for hMBPH (Fig. 3). Fig.
3A shows a typical elution profile of an eosinophil granule
preparation fractionated over a Sephadex G-50 column (29, 31).
Fractions 71-90 reacted with J191-12H11 by Western blotting (data not
shown) and were pooled and refractionated over a second Sephadex G-50
column (Fig. 3B). Samples from even numbered fractions
54-80 from this second gel filtration column were electrophoresed on
three SDS-polyacrylamide gels. A Western blot of one gel was tested for
hMBP (Fig. 3C), and another was tested for hMBPH (Fig.
3D). The third gel was stained with Coomassie Brilliant Blue
R-250 (Fig. 3E). Note the apparent shift from the slightly
higher molecular weight bands of hMBP (fractions 54-64) to the lower
molecular weight bands of hMBPH (fractions 64-80) in Fig.
3E consistent with their respective calculated molecular
weights (Table IV) and the position of
the trough (fractions 63 and 64) separating the two elution peaks in
Fig. 3B. Fraction 64 in Fig. 3E best shows the
presence of two bands. Similar elution profiles have been obtained when
using eosinophil granule preparations from the four hypereosinophilic syndrome patients tested.
To verify the identities of the proteins composing the two peaks from
the second Sephadex G-50 column (Fig. 3B), fraction 57 from
the ascending side of peak 1 and fraction 76 from the descending side
of peak 2 were subjected to N-terminal amino acid sequencing. As shown
in Fig. 4, the resulting amino acid
sequences corresponded to those anticipated for the N termini of hMBP
(41-43) and hMBPH. Similarly, the molecular masses determined by mass spectrometry for proteins from the ascending side of peak 1 and from
the descending side of peak 2 were in general agreement with the
molecular masses calculated from the primary sequences for hMBP and
hMBPH, respectively (Table IV). In both cases, however, the measured
molecular mass was approximately 5 Da lower than predicted. The
presence of two disulfide bonds in hMBP had previously been reported
(44). Thus, disulfide linkages were suspected as the cause for the
reduced experimental molecular masses. Each such disulfide linkage
results in the loss of two hydrogen atoms with a concurrent reduction
in molecular mass of 2 Da; however, the mass accuracy obtained was
insufficient to determine if two or three such linkages were present.
Therefore, two further experiments were performed for each protein.
Initially, the proteins were alkylated with IA and then examined by
mass spectrometry (Table IV). This provided the number of reactive
cysteines present within each protein (Table IV). As expected,
nonreduced hMBP contained five reactive cysteines, whereas hMBPH
contained six reactive cysteines. To provide the number of disulfide
bonds within each protein, fresh protein samples were reduced with
dithiothreitol and alkylated with IA (Table IV). These mass
spectrometry data confirmed the presence of two disulfide linkages in
hMBP and suggested an identical number in hMBPH.
Extinction Coefficients of Human Major Basic Proteins--
To
allow accurate concentration estimates of protein used in biological
assays, the extinction coefficients of hMBP and hMBPH were determined
(see "Experimental Procedures"). Experimental determinations
yielded extinction coefficients at 280 nm of 3.67 ± 0.09 and
3.32 ± 0.04 (mg/ml) Cytotoxic and Cytostimulatory Properties of hMBPH--
As a
measure of cytotoxicity, inhibition of protein synthesis in K562 cells
was monitored via [14C]leucine incorporation3
after incubation with a human MBP or the known cytotoxin, melittin. Fig. 5 shows the concentration dependence
of protein synthesis inhibition by hMBP and hMBPH. While both proteins
inhibited cellular protein synthesis in the micromolar range, an
approximately 3 times greater concentration of hMBPH was necessary to
inhibit comparably with hMBP. Potential synergism of hMBP and hMBPH was also tested (data not shown). Samples containing 1 µM
hMBP plus 1 µM hMBPH or 2 µM hMBP plus 1 µM hMBPH resulted in additive cytotoxicity. Thus, no
synergism was observed.
Fig. 5 also shows the cytotoxic effects of other human eosinophil
granule proteins. Eosinophil cationic protein (ECP) and EDN are both
RNases with molecular masses of approximately 19 kDa by
SDS-polyacrylamide gel electrophoresis and with calculated pIs near
10.9 and 8.9, respectively (3). Eosinophil peroxidase has a molecular
mass of approximately 70 kDa and a calculated pI near 10.9 (3).
Interestingly, these highly basic proteins, ECP and eosinophil
peroxidase, also inhibited protein synthesis. ECP cytotoxicity was
pronounced even at 1 µM, but unexpectedly plateaued at
approximately 50% inhibition. The cytotoxicity of ECP was eventually
lost at a concentration of 0.1 µM (data not shown). Only
EDN, which has a similar mass and nearly identical calculated pI and
net positive charge (7+) to that of hMBPH (Table I), showed minimal
inhibition of protein synthesis. This result suggests that a positive
charge per mass unit ratio similar to that of hMBPH is not sufficient
for cytotoxicity. This interpretation, however, is obscured by the lack
of precise characterization of the charge contribution resulting from
EDN glycosylation (46, 47).
Human MBP also exhibits a variety of cytostimulatory activities (3).
Fig. 6 compares the ability of hMBP and
hMBPH to stimulate those cellular responses. Human MBP and hMBPH
stimulated neutrophil superoxide production virtually identically (Fig.
6A). In contrast, hMBPH stimulated neutrophil IL-8
production (Fig. 6B) about half as potently as hMBP, but still
comparable with that of lipopolysaccharide. Likewise, concentrations of
hMBPH approximately 4- and 10-fold greater than that of hMBP were
required to stimulate comparable histamine (Fig. 6C) and
leukotriene C4 (LTC4; Fig. 6D)
release from basophils, respectively. Each of these appears to involve an active cellular process and is not a result of cytolysis (11, 13,
35, 48).2 However, it appears that hMBP is inhibitory to
de novo LTC4 production when present at 4 µM (Fig. 6D).
Previously our laboratory has characterized the principal proteins
contained within the human eosinophil granule (1, 2). To search for
novel eosinophil proteins we stimulated UCC with IL-5 and sequenced
15,000 plasmid cDNA inserts. The result of this analysis, referred
to as a Transcript Image, revealed a second prepro-MBP-like gene
expressed by the human IL-5-differentiated UCC. Knowledge of the
cDNA sequence for this hprepro-MBP homolog allowed analysis of its
tissue expression profile; its expression was detected in bone marrow
only. The cDNA sequence also allowed production of a monoclonal
antibody to the corresponding protein homolog in eosinophil granules.
Purification of hMBPH protein permitted comparison of its activity with
that of hMBP in a series of cytotoxicity and cytostimulatory assays.
These data showed qualitatively similar activities between hMBPH and
hMBP but with diminished potency for the homolog in the majority of
assays. Thus, the Transcript Image approach to quantitative and
qualitative assessment of gene transcript expression successfully
identified and led to the isolation of a novel eosinophil granule protein.
Transcript Imaging--
The sequencing of approximately 10,000 clones, as in current Transcript Imaging, permits random identification
of many known and novel rare messages, with a cumulative identification
of rare messages for each additional sample studied. Furthermore, this procedure can detect either increased or decreased levels of gene expression that can later be compared with or reanalyzed in light of
subsequent sequence data. The relatively long sequences from Transcript
Imaging, often in the coding region and not subject to possible PCR
anomalies, can also allow for polymorphism/mutation analyses. Moreover,
the sequenced cDNA is in a useable form, i.e. within the
multiple cloning site of a plasmid. However, sequencing 10,000 clones
still remains approximately 10-100-fold below the level necessary to
achieve a quantitative gene expression profile that includes most rare
messages (0.01% or less of a cell's total mRNA) (19, 49, 50).
Other current approaches to gene discovery and/or differential gene
expression analysis include differential display (51), suppression
subtractive hybridization (52), microarray hybridization (53), and
serial analysis of gene expression (54). Each technique ultimately has
advantages and disadvantages. Limitations of Transcript Imaging include
the requirement for a substantial quantity of starting polyadenylated
mRNA, approximately 2 µg, and the extensive labor and cost
involved in generating sequence data for thousands of cDNA clones.
However, of the above mentioned methods only differential display (51)
requires substantially less RNA, but with a corresponding loss in
completeness of the gene expression profile. Alternatively, serial
analysis of gene expression (50, 54, 55) may be a viable option to
generate a comparable gene expression profile of both known and unknown
genes with less labor and cost. However, with serial analysis of gene
expression, further work to generate a more complete cDNA sequence
may be necessary. Similarly, with increasing knowledge of human
cDNA and genomic DNA sequences, microarray hybridization technology
(53) may allow screening of whole genomes if the appropriate
complementary sequences are known and available. Nonetheless,
Transcript Image technology remains a valuable approach to both
quantitative and qualitative assessment of gene expression in cells or tissues.
Protein Sequence Comparisons of Major Basic Proteins--
Other
species known to produce a protein homologous to hMBP include mouse,
rat, and guinea pig (reviewed in Ref.
1).4 Interestingly, guinea
pigs produce two distinct MBP homologs of similar molecular weights and
isoelectric points (calculated pI values of 11.7 and 11.3) (56). The
presence of a second MBP gene in the mouse has also been proposed (57).
Identification of a homolog of hMBP brings the number of known
mammalian prepro-MBP sequences to six. However, the uniqueness of
hprepro-MBPH becomes apparent upon alignment of these sequences (Fig.
7).
In the signal sequence, only six amino acids are conserved throughout
all the known MBPs. However, the more general signal sequence pattern
of one or more positively charged amino acids followed by a continuous
stretch of 6-12 hydrophobic amino acids (58) is present in
hprepro-MBPH as in the other forms. Therefore, it is likely, and its
presence in eosinophil granule preparations and preliminary protein
expression data5 confirm that
hMBPH can also be secreted.
In the pro section, even less identity is observed across the various
homologs (Fig. 7). However, the total number of acidic residues (Asp or
Glu) for each of the pro sections remains in the range of 25-32, with
hMBPH's pro section containing 28 (Table I). Unlike the abundant
negative charge, none of the six glycosylation sites determined for
hpro-MBP (Fig. 7) (59) appear to be strictly retained in hpro-MBPH.
Alternative O-linkage sites (Ser and Thr) near the
N-terminal of the pro section of hpro-MBPH could potentially substitute
for those O-linked sites glycosylated in hpro-MBP. Likewise,
the serine at position 79 carrying the O-glycosaminoglycan is present in hpro-MBPH, but its two nearest C-terminal side amino acids do not conform as well with the Ser-Gly-Ser sequence (60, 61) at
or near this position in all other pro-MBPs. This lack of conserved
glycosylation sites is consistent with our preliminary data indicating
that hpro-MBPH expressed in Chinese hamster ovary cells is secreted in
an unglycosylated form.5 Of note, the fully
glycosylated serine at position 25, like the O-glycosaminoglycan site mentioned above, is the only other
hpro-MBP glycosylation site conserved in all previously known pro-MBPs. Therefore, it appears that hpro-MBPH is substantially less glycosylated than any of the other homologs. The possible consequence(s) of this
remains to be determined, but in general glycosylation can be involved
in protein trafficking, protein folding, resistance to proteolysis, and
molecular recognition/binding.
In the MBP section, the greatest degree of homology is found with 42 of
the approximately 117 amino acids being identical. However, in contrast
to the pro section of hpro-MBPH, where homology is low but net negative
charge is conserved, the highly homologous MBP section of hMBPH has a
net positive charge, which is substantially diminished compared with
the other MBPs. Specifically, the net charge of hMBPH at neutral pH is
near 8+ while hMBP and the other MBPs have about 16+ to 20+ net charge
(Table I and Fig. 7). However, strict conservation of most basic amino
acids appears to be of minimal importance. With inclusion of hMBPH in
the amino acid sequence alignment, only a single basic residue,
arginine at position 234, is strictly conserved. Moreover, even without
inclusion of hMBPH in the alignment, only an additional two basic
residues, arginine at positions 154 and 241, are conserved among the
remaining five MBPs. Overall, with regard to basicity, hMBPH appears to be the most divergent form of the known MBPs.
The 2-fold reduction in net positive charge of hMBPH appears to reduce
its ability to inhibit cellular protein synthesis, to induce neutrophil
IL-8 production, and to induce basophil histamine and LTC4
release compared with hMBP (Figs. 5 and 6). The more basic granule
proteins, ECP and eosinophil peroxidase, also demonstrated greater
protein synthesis inhibition than the less basic EDN. This suggests the
importance of a molecule's net positive charge in this cytotoxicity
assay. However, arguing against net positive charge as the sole
determinant of the hMBP's activities are the following: 1) comparable
induction of neutrophil superoxide production by hMBP and hMBPH (Fig.
6); 2) the lack of cytotoxicity by EDN (Fig. 5), a molecule of similar
mass and apparently similar net charge to hMBPH; and 3) the inability
of ionic strength equivalent to 0.125 M to markedly
diminish hMBP's interaction with
1,2-dimyristoyl-sn-glycero-3-phosphocholine:1,2-dimyristoyl-sn-glycero-3-phosphatidic acid or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl
serine membranes or its bactericidal activity (62).
Interestingly, the negatively charged aspartic acid at position 200 is
conserved throughout all MBPs and is the only acidic amino acid in two
of the six MBPs. Perhaps the arginine at position 234 and the aspartic
acid at position 200 form a critical ion pair. The vast majority of the
remaining conserved residues have low or no polarity including 11 aromatic and seven glycine residues. In addition to their probable role
in protein folding, the functional importance of hydrophobic residues
may be reflected by the above mentioned lack of influence of ionic
strength changes on some hMBP activities and by the general amphipathic
nature of several toxic, cationic polypeptides (33). Notably, the
abundance of aromatic residues in the human MBPs also results in their
extremely high extinction coefficient values (45).
With regard to the two aforementioned disulfide linkages, the reactive
half-cystine residues of hMBP (44) are conserved in hMBPH and in all
other known homologs. Thus, these protein folding elements appear to be
conserved in hMBPH as well. Two of the 3-6 remaining free sulfhydryls
are also conserved among the homologs. Reduction and alkylation of hMBP
does influence some of its apparent biological activities (10, 13, 48). Therefore, these cysteine residues appear to have biological
importance, although how they contribute to MBP effector function is obscure.
The sequence similarities among hMBPH, hMBP, and the other MBPs,
suggest a gene duplication event. Presumably, this gene duplication occurred early in mammalian evolution, because guinea pigs, and possibly mice, have two prepro-MBP genes. However, both forms of guinea
pig MBP have a characteristically high pI, in contrast to hMBPH of the
human MBPs. Therefore, hMBPH appears to either be evolving toward a new
function or drifting away from a previous function (38).
The level of transcription of hprepro-MBPH was 14% of that for
hprepro-MBP but still 1.1% of the total inserts sequenced for Transcript Imaging of IL-5 differentiated UCC. Similarly, the quantity
of hMBPH protein isolated from eosinophil granules was a small
percentage relative to hMBP. However, while hMBPH expression may not
equal that of hMBP, it still may be present at relevant quantities
within the human eosinophil. Current efforts are under way to more
precisely localize and quantitate hMBPH protein in human eosinophils
and tissues. This may allow hMBPH to function as an additional marker
for the human eosinophil.
In summary, the identification of hMBPH via Transcript Imaging has
verified the presence of a second MBP-like molecule in humans, as in
guinea pigs. In general, the pre, pro, and MBPH sections and the two
disulfide linkages within the MBP section are conserved. The tissue
expression profile of hprepro-MBPH is similar to that of hprepro-MBP,
except for the expression of hprepro-MBP by the placenta. Human MBPH,
however, is much less basic than the other known MBPs and this tended
to diminish, but not abolish, hMBPH's activity in cytotoxicity and
cytostimulatory assays as compared with hMBP. Finally, the isolation of
a second human MBP gene and protein will allow assessment of
comparative gene promoter structure (39), evolution, protein processing
and cellular localization, and protein structure-function relationships
between the human eosinophil granule MBPs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until
processing. Total RNA was extracted using standard CsCl methods (24).
One hundred micrograms of pooled total RNA was utilized for
construction of a cDNA library for Transcript Image generation.
20 °C
as purified hMBPH.
) for hMBPH and hMBP were
also calculated using the equation
(M
1
cm
1) = 5540(nTrp) + 1480(nTyr) + 134(nS-S)
(32), where nTrp, nTyr,
and nS-S are the numbers of tryptophans,
tyrosines, and disulfide linkages within the protein of interest
(hMBPH: 7 Trp, 5 Tyr, and 2 S-S; hMBP: 7 Trp, 6 Tyr, and 2 S-S).
-radiation.
incorporation with test protein/incorporation with buffer
only) × 100.
2) release was measured as nmol of ferricytochrome C reduced/106 neutrophils/30 min as
described previously (35). Spontaneous superoxide anion production was
from cells incubated without stimulus.
20 °C until measurement of IL-8 content using a specific
enzyme-linked immunosorbent assay (BIOSOURCE
International, Camarillo, CA). Spontaneous IL-8 production was from
cells incubated without stimulus.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
23 most abundant transcripts of IL-5-differentiated UCC
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Fig. 1.
cDNA and deduced amino acid sequences of
human prepro-MBP homolog. This consensus cDNA sequence of
hprepro-MBPH was determined by sequencing multiple clones generated
during the Transcript Image procedure. The MacVector 4.5 program
(Kodak) was used to deduce the amino acid sequence from the cDNA
codons. The start of the "pro" ( ) (59) and "MBP" (
)
sections are marked. Nucleotide sequences corresponding to the
annealing positions of the hprepro-MBPH-specific PCR primers are
underlined. The amino acid sequence of the peptide used in
J191-12H11 monoclonal antibody production is underlined.
The asterisk indicates the stop codon.
Gross structural and ionic comparison of human prepro-MBP and
prepro-MBPH
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Fig. 2.
Tissue localization of hprepro-MBP and
hprepro-MBPH message. A, the specificity of the
hprepro-MBP (left) and hprepro-MBPH (right)
primers are shown using water (Water), a plasmid containing
hprepro-MBP cDNA (proMBP), and a plasmid containing
hprepro-MBPH cDNA (proMBPH) as template in PCRs as
described under "Experimental Procedures." The positions of DNA
markers (Markers) are indicated by the lines on
the left side of A, from
top to bottom: 310, 271/281, 234, 194, 118, and
72 nucleotides in length. hprepro-MBP primers (B) and
hprepro-MBPH primers (C) were used with the first-strand
cDNA from the various tissues indicated (bone marrow
(BM), lymph node (LN), peripheral blood
leukocytes (PBL), pancreas (Panc.), placenta
(Plac.), prostate (Pros.), skeletal muscle
(SM), small intestine (SI)) in PCRs as described
under "Experimental Procedures." A list of all tissues tested for
hprepro-MBP and hprepro-MBPH is provided under "Experimental
Procedures." C also shows a negative (Water)
and positive (proMBPH) control as in A.
Electronic Northern tissue expression profile of human prepro-MBPH
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Fig. 3.
Isolation of hMBPH from eosinophil
granules. A shows a Sephadex G-50 column elution
profile of an eosinophil granule preparation (see "Experimental
Procedures"). The two lines intersecting the
elution profile in A bracket fractions 71-90. B
shows an elution profile of fractions 71-90 (from A), which
were pooled, concentrated, and rechromatographed on a second Sephadex
G-50 column. Even-numbered fractions 54-80 were analyzed by
SDS-polyacrylamide gel electrophoresis, 15%, on three separate gels.
C shows the Western blot analysis using the hMBP-specific
monoclonal antibody, J6-8A4, and D shows the analysis using
the hMBPH-specific monoclonal antibody, J191-12H11. E shows
the third gel stained with Coomassie Brilliant Blue R-250. Positions of
protein molecular mass markers (top to bottom),
21.5 and 14.4 kDa in C and D and 97.4, 66.2, 43, 31, 21.5, and 14.4 kDa in E, are indicated by the
lines at the left in each panel.
Electrospray ionization mass spectrometric analysis of human MBP and
MBPH
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Fig. 4.
N-terminal amino acid sequencing of hMBP and
hMBPH. A portion of fraction number 57 (representing peak 1) and
76 (representing peak 2) from the second Sephadex G-50 column (Fig.
3B) were analyzed by Edman degradation microsequencing. The
resulting N-terminal amino acid sequences are shown.
1 cm
1 for hMBP and
hMBPH, respectively. While these values are high compared with other
proteins (45), they are in good agreement with theoretically calculated
extinction coefficients of 3.47 and 3.46 (mg/ml)
1
cm
1 for hMBP and hMBPH, respectively, based on
tryptophan, tyrosine, and disulfide linkage content.
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Fig. 5.
Inhibition of cellular protein synthesis by
eosinophil granule proteins. Leucine-starved K562 cells growing in
log phase were exposed to buffer alone and 1 µM
(open bars), 3.3 µM
(hatched bars), and 10 µM
(closed bars) of the test agent listed on the
abscissa for 2 h followed by the addition of 0.1 µM [14C]leucine. Cyclohex.,
cycloheximide. After a final incubation of 2 h, total protein
incorporation of [14C]leucine was assessed and compared
with that in the presence of buffer only. The percentage of inhibition
of [14C]leucine incorporation was calculated as described
under "Experimental Procedures." Each bar represents the
mean percentage of inhibition calculated from two separate experiments,
where each test agent was tested in triplicate at each of the three
concentrations tested. S.D. values are shown by the error
bars or are indistinguishable from the top of the
data bars.
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Fig. 6.
Comparison of hMBP and hMBPH cytostimulatory
effects. A, neutrophils from four donors were incubated
with the indicated concentrations of hMBP (closed
squares) or hMBPH (closed circles) for
30 min at 37 °C, and superoxide anion production was quantified as
described under "Experimental Procedures." Results are the
mean ± S.D. after subtraction of spontaneous production (1.4 ± 1.7 nmol/106 cells/30 min). B, neutrophils
were incubated with sodium acetate vehicle buffer (NaOAc), hMBP (2 µM), hMBPH (2 µM), or lipopolysaccharide
(LPS, 100 ng/ml) for 20 h at 37 °C, and IL-8 release
was measured as described under "Experimental Procedures."
Neutrophils from four donors were tested, and each mean ± S.D.
after subtraction of spontaneous release (0.04 ± 0.04 ng/ml) is
shown. C, purified basophils (59-88%) were incubated with
the indicated concentrations of hMBP (closed
squares) and hMBPH (closed circles)
for 30 min at 37 °C, and histamine release was quantified as
described under "Experimental Procedures." Cells from three donors
were tested, and each value is the mean ± S.D. after correction
for spontaneous histamine release (9 ± 3%). D,
purified basophils were incubated with the indicated concentrations of
hMBP (closed squares) and hMBPH
(closed circles) for 30 min at 37 °C, and
LTC4 release was quantified as described under
"Experimental Procedures." Cells from three donors were tested, and
each value is the mean ± S.D.; spontaneous LTC4
release was undetectable. The values shown as 0 µM
protein concentration in A, C, and D
represent production or release due to the sodium acetate vehicle
buffer for hMBP and hMBPH. Error bars not visible
in the graphs are contained within the dimensions of the
data symbol.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Alignment of prepro-MBPs to hprepro-MBP
homolog. MacVector 4.5 (Kodak) was used to align the six known
prepro-MBP amino acid sequences. Identical amino acids among the
prepro-MBPs are enclosed in boxes. Half-cystines as
determined for hMBP (44) are shaded with gray.
Partially substituted O-linked (closed
triangles), fully substituted O-linked
(closed diamonds), N-linked
(closed square), and glycosaminoglycan
(closed rectangle) glycosylation sites as
reported by Oxvig et al. (59) are shown. The start of
pro-MBP ( ) and MBP (
) are also marked. Accession numbers for the
sequences shown are listed in the Footnotes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Cris Seminario for procuring umbilical cord blood, Doug Gooding (Incyte Pharmaceuticals) for cDNA library construction, Andrew Schimming for assisting with the design of PCR primers, and Cheryl Adolphson and Linda Arneson for assisting with manuscript preparation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI 32041 (to L. L. T.) and AI 09728, AI 34577, and AI 07047 (to G. J. G.) and by the Mayo Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 132209.
§ To whom correspondence should be addressed: Dept. of Immunology, Mayo Clinic, Rochester, MN 55905. E-mail: plager.douglas{at}mayo.edu.
2 Page, S. M., Gleich, G. J., and Thomas, L. L. (1999) J. Immunol. Methods, in press.
3 H. Kubo, D. A. Loegering, Y. Tohda, J. Bankers-Fulbright, C. R. Weiler, H. Nakajima, L. L. Thomas, C. R. Adolphson, and G. J. Gleich, submitted for publication.
4 The deduced amino acid sequence of hprepro-MBPH has been scanned against the nonredundant GenBankTM/Protein Data Bank/Swiss-Prot/PIR data base, and the following sequences were found to be significantly homologous: human eosinophil granule major basic protein precursor, gi 119239, sp P13727, or Y00809; mouse major basic protein, gi 1109659 or L46768; guinea pig eosinophil granule major basic protein 1 precursor, gi 119238, sp P22032, or D90251; guinea pig eosinophil granule major basic protein 2 precursor, gi 544241, sp P35709, or D00817; rat eosinophil major basic protein precursor gi 2143721, pir S68150, or D50568; similarity to C-type lectin domains, gi 1326393 or U58752.
5 D. A. Plager and G. J. Gleich, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MBP, major basic protein; hMBP, human major basic protein; hMBPH, human major basic protein homolog; hprepro-MBP, human prepro-MBP; hprepro-MBPH, human prepro-MBPH; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; IA, iodoacetamide; IL, interleukin; UCC, umbilical cord precursor cells; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; LTC4, leukotriene C4.
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