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(Journal of Leukocyte Biology. 2002;72:478-485.)
© 2002 by Society for Leukocyte Biology

Molecular characterization and expression analysis of leucine-rich {alpha}2-glycoprotein, a novel marker of granulocytic differentiation

Lynn C. O’Donnell, Lawrence J. Druhan and Belinda R. Avalos

Bone Marrow Transplant Program, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University College of Medicine and Public Health, Columbus

Correspondence: Dr. Belinda R. Avalos, The Ohio State University, Bone Marrow Transplant Program, A437A Starling-Loving Hall, 320 West Tenth Avenue, Columbus, OH 43210. E-mail: avalos-1{at}medctr.osu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using data obtained from cDNA representational difference analysis to identify genes induced during neutrophilic differentiation of the 32D clone 3G (32Dcl3G) cells, we isolated cDNA clones for murine and human leucine-rich {alpha}2-glycoprotein (hLRG), a protein with unknown function purified 25 years ago. Expression of LRG during differentiation of 32Dcl3G cells preceded the expression of lactoferrin and gelatinase but followed myeloperoxidase. LRG transcripts were also detected in human neutrophils and progenitor cells but not in peripheral blood mononuclear cells. Notably, LRG expression was up-regulated during neutrophilic differentiation of human MPD and HL-60 cells but down-regulated during monocytic differentiation of HL-60 cells. The hLRG gene was localized to chromosome 19p13.3, a region to which the genes for several neutrophil granule enzymes also map. The putative promoter region of LRG was found to contain consensus-binding sites for PU.1, C/EBP, STAT, and MZF1. These results suggest that LRG is a novel marker for early neutrophilic granulocyte differentiation.

Key Words: RDA • 32Dcl3G • LRG • myelopoiesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leucine-rich {alpha}2-glycoprotein (LRG) was first identified as a trace protein in human serum in 1977 [1 ]. The primary sequence of LRG was determined using Edman degradation, which revealed a marked periodicity in the leucine residues in this protein [2 ]. At least eight repeating 24 amino acid segments with a notable consensus sequence were identified in LRG. This 24 amino acid consensus sequence, termed the leucine-rich repeat (LRR), has since been identified in a large family of proteins [3 , 4 ]. Although the functions of many of the members of the LRR-containing superfamily are known, the function of LRG has not been elucidated.

Considerable efforts have focused on the mechanisms by which granulocyte-colony stimulating factor (G-CSF) induces granulocytic differentiation [5 ]. The murine interleukin (mIL)-3-dependent 32D clone 3G (32Dcl3G) cell line was established from normal murine diploid bone marrow cells and differentiates into mature neutrophils in response to G-CSF [6 ]. This cell line has been used as a model system for investigating G-CSF-induced differentiation. To identify genes induced by G-CSF during neutrophilic granulocyte differentiation, we used cDNA representational difference analysis (RDA) to generate a representational cDNA library enriched for neutrophil-specific transcripts from G-CSF-treated 32Dcl3G cells. Further characterization of one of the positive clones obtained using this method revealed its identity as LRG. We report here the identification of mLRG and human LRG (hLRG) cDNA clones and the genomic sequences of the LRG genes, the structures of these genes, and their chromosomal localization. Additionally, we show that expression of LRG is induced early during granulocytic differentiation and persists through the neutrophil stage. Expression of LRG in myeloid cells appears to be specific for the neutrophilic granulocyte lineage, as no expression of LRG could be detected in primary human monocytes or cell lines induced to differentiate along the monocyte pathway. G-CSF and chemical inducers of neutrophilic differentiation up-regulated the expression of LRG, indicating that this effect is not unique to the G-CSF pathway. These results suggest that LRG is a novel marker for neutrophilic granulocyte differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purified recombinant human (rh)G-CSF was generously provided by Amgen Inc. (Thousand Oaks, CA). G-CSF responsive murine 32Dcl3G cells were kindly provided by Dr. Giovanni Rovera (The Wistar Institute, Philadelphia, PA); the human promyelocytic leukemia HL-60 cell line, by Dr. Jas Lang (The Ohio State University, Columbus); the G-CSF responsive human MPD cell line derived from a patient with a myeloproliferative disorder, by Drs. Michael Baumann and Cassandra Paul (Wright State University, Dayton, OH); and WEHI-3B cells, by Dr. Harvey Lodish (Massachusetts Institute of Technology, Cambridge).

Cells
All cell lines were maintained in RPMI-1640 medium (Gibco-BRL, Grand, Island, NY), supplemented with 10% v/v fetal bovine serum, 2 mM glutamine, and antibiotics. IL-3-dependent 32Dcl3G cells also required the presence of conditioned medium from WEHI 3B cells (10% v/v) as a source of IL-3 [7 ]. The 32Dcl3G cells were washed twice with phosphate-buffered saline to remove IL-3 before transfer to medium containing rhG-CSF (10 ng/mL) to induce neutrophilic granulocyte differentiation. Granulocytic differentiation of HL-60 cells was initiated by the addition of dimethyl sulfoxide (DMSO; 1.25% v/v) to the culture medium [8 ]. For monocytic differentiation of HL-60 cells, phorbol 12-myristate 13-acetate (PMA; 5 nM) was added to the culture medium [9 ]. For the maintenance of MPD cells, sodium pyruvate was added to the medium to a final concentration of 1 mM. Neutrophilic granulocyte differentiation of MPD cells was initiated by the addition of rhG-CSF (2.85 ng/mL) [10 ]. Differentiation of all cell lines was confirmed by morphology using Wright-Giemsa staining and reverse transcriptase-polymerase chain reaction (RT-PCR; see below) for expression of the neutrophil markers myeloperoxidase (MPO), lactoferrin (LF), and gelatinase (GEL) or by nitroblue tetrazolium (NBT) assays [11 ].

Peripheral blood (PB) and bone marrow (BM) were obtained from hematologically normal donors following informed consent and were collected in EDTA-containing tubes. Mononuclear cell fractions were isolated using density gradient centrifugation with Histopaque 1.077 (Sigma Chemical Co., St. Louis, MO). Neutrophils were isolated from the resulting cell pellet using dextran sulfate (3% w/v) sedimentation and hypotonic lysis of red blood cells. Wright-Giemsa staining was used to confirm >97% purity of the neutrophils, which were used for subsequent analysis.

cDNA RDA
To identify genes induced during G-CSF-stimulated differentiation, RNA was extracted from 32Dcl3G cells grown in the presence of IL-3 or in the presence of rhG-CSF. TrizolTM reagent (Gibco-BRL) was used to extract total RNA from four different populations of 32Dcl3G cells: those grown in the presence of IL-3 for 5 days (designated I5) and 7 days (I7) and those grown in the presence of rhG-CSF for 5 days (G5) and 7 days (G7). Poly-A RNA was isolated from each total RNA preparation using the PolyATtract system (Promega, Madison, WI). The resultant mRNA preparations were treated with DNase I, phenol/chloroform-extracted, ethanol-precipitated, and resuspended in water. For the preparation of cDNA, 5 µg from the 5 day and 7 day mRNA preparations for each cytokine (I5+I7 and G5+G7) were pooled and diluted to 0.4 µg/µL. The pooled samples were then used to produce double-stranded cDNA using the cDNA Synthesis System kit (Gibco-BRL) according to the manufacturer’s instructions.

In preparation for cDNA RDA, the double-stranded cDNAs were precipitated twice at -20°C with 1/5 vol 10 M ammonium acetate and an equal volume of isopropanol and were resuspended in TE. Using the technique described by Hubank and Schatz [12 ], we used 2 µg each pooled cDNA in the preparation of separate driver and tester representational cDNA populations. The driver cDNA was from IL-3-treated cells (I5+I7), and the tester cDNA was from G-CSF-treated cells (G5+G7). RDA was then performed using driver cDNA hybridized in excess against tester cDNA under conditions where subsequent PCR reactions subtracted sequences common to both populations and amplified sequences unique to the tester.

After three rounds of hybridization and subtraction, the heterogeneous final difference product (DP3) was digested with DpnII, cloned into the BamHI site of the pBluescript KS+ phagemid (Stratagene, San Diego, CA), and transformed into DH5{alpha} Escherichia coli. Each clone contained a DpnII cDNA fragment from a population enriched for G-CSF-induced transcripts. cDNA inserts from individual clones were then PCR-amplified and screened for differential expression by dot blot analysis using gel-purified driver DP3 and tester DP3 as probes. This initial screen confirmed that 76 of 141 RDA clones contained cDNA inserts enriched in the tester DP3. For clones that were found to be more abundant in the tester DP3 by dot blot, differential expression was subsequently confirmed by Northern blot analysis of the original total RNA samples. The differentially expressed RDA clones were then sequenced and compared with the public nucleotide and protein databases using BLAST [13 ].

Identification and characterization of cDNAs and genomic sequences corresponding to differentially expressed RDA clones
BLAST queries of the mouse and human expressed sequence tag (EST) databases were used to identify putative full-length cDNA clones of differentially expressed RDA clones. Additional cDNA clones were identified in UniGene clusters that contained the EST clones of interest. Candidate EST clones were purchased from Genome Systems Inc. (Incyte Genomics, Inc., Palo Alto, CA) or Research Genetics Inc. (Invitrogen, Carlsbad, CA) and were analyzed by restriction analysis and end-sequencing. Both strands of the clones most likely to contain the full-length cDNA of interest were sequenced. The cDNA sequences were scanned for open reading frames (ORF) using MacVector (Eastman Kodak, Rochester, NY). The putative ORFs were analyzed for the presence of signal peptides using the algorithms SignalP V2.0 and TargetP V1.0 [14 15 16 ].

Genomic sequences corresponding to the identified full-length cDNA clones were identified by BLAST queries of GenBank, the mouse genome database, or the Celera Inc. (Rockville, MD) human genome database [17 18 19 ]. Chromosomal assignments were based on the published physical location of the identified genomic clone or by analysis of the genomic sequence with electronic PCR [20 ]. The intron/exo structures of the identified genomic sequences were determined by alignment of the full-length cDNA to the genomic sequence using BLAST2 [21 ]. Further analysis of the genomic sequences using McPromoter V3b and the Berkeley Drosophila Genome Project (BDGP) Neural Network Promoter Prediction algorithms (University of California, Berkeley) was done to identify putative transcription start sites [22 , 23 ]. Putative promoter regions were scanned against the TRANSFAC database using MatInspector Professional (Genomatix, München, Germany) to identify putative binding sites for transcription factors [24 , 25 ]. Genomic sequences were compared within the 1000-bp region upstream of each of the identified ORFs by Pustell DNA dot matrix analysis using a window-sized setting of 20, minimum homology score of 65%, and hash value of 4 [26 ].

Northern blot hybridization
Total RNAs (10 µg each) were irreversibly denatured with glyoxal/DMSO and were then size-fractionated on a 1% w/v agarose gel [27 ]. RNA was transferred to charged nylon membranes and hybridized with murine- or human-specific probes. A 537-bp murine-specific LRG probe was prepared by liberation of the insert from the G11 RDA clone with EcoRI and NotI. A 395-bp, human-specific LRG probe was separated from IMAGE clone 81861 that had been digested with BstXI and EcoRI. This probe is common to both classes of hLRG transcripts. To prepare a class II-specific hLRG probe, the 3' end of IMAGE clone 85213 was PCR-amplified using a primer specific for the 3' end of the class II LRG transcript (5'-GCTTCCTAGAACACACGATG-3') and a primer specific for the LacZ portion of the vector (5'-CCCAGTCACGACGTTGTAAAACG-3'). The amplified product was digested with XhoI to remove the vector sequence, yielding a 295-bp fragment containing sequence specific for the class II hLRG. All probes were gel-purified prior to labeling using the QIAquick gel extraction kit (Qiagen, Chatsworth, CA). Probes were labeled with 32P using the High Prime kit (Roche/BMB, Nutley, NJ) and were purified using size-exclusion spin columns. Hybridization and washing were performed with ExpressHyb (Clontech, Palo Alto, CA) hybridization solution according to the manufacturer’s instructions. Blots were exposed to X-ray film or a phosphor-imaging screen (Molecular Dynamics, Sunnyvale, CA). Northern blot quantitation was performed using the Imagequant software (Molecular Dynamics). The mouse multiple tissue Northern blot used was purchased from Clontech.

RT-PCR
Reverse transcription was carried out on 2 µg total RNA from each sample using SuperscriptTM RT (Gibco-BRL) and random hexamer primers according to the manufacturer’s instructions. PCR with gene-specific primers was performed using Taq DNA polymerase (Gibco-BRL) at a final MgCl2 concentration of 2 mM. The PCR products were size-fractionated on agarose/TAE gels (1% w/v) and visualized with ethidium bromide staining. The thermocycling program for all reactions included an initial incubation at 94°C for 5 min, followed by 30 cycles of denaturation (94°C, 30 s), annealing (see temperatures below, 30 s), and extension (72°C, 30 s), with a final incubation at 72°C for 7 min. PCR with the mMPO-specific primers murine MPOfor (5'-AACCAGCTGGGGCTGCTGGCTGTCAATACACG-3') and mMPOrev (5'-AACTCCAGGTTCTTCAGCACCGTGCCG-3') was performed at an annealing temperature of 62°C, which resulted in an 806-bp product. PCR with the murine LF-specific primers mLFfor (5'-GCCAGTCACAGGAGAAGTTTGG-3') and mLFrev (5'-GCCATTGCTTTTGGAGGATTTC-3') was performed at an annealing temperature of 54°C, resulting in a 452-bp product. PCR with the murine GEL-specific primers mGELfor (5'-ACAACTGAACCACAGCCGACAG-3') and mGELrev (5'-TCATTTTGGAAACTCACACGCC-3') was performed with an annealing temperature of 54°C, producing a 743-bp product.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of differentially expressed LRG clones
We initially examined the time course for G-CSF-induced morphologic changes in 32Dcl3G cells to optimize our chances of isolating genes that are induced early during neutrophilic granulocyte differentiation. G-CSF treatment of 32Dcl3G cells resulted in a decrease in the blast cell population from 90% at day 0 to 10% at day 14 (Fig. 1 ). The presence of mature neutrophils could be detected by day 5, at a time when more than 50% of the cells had differentiated into myelocytes and metamyelocytes. With continued growth in G-CSF, the numbers of myelocytes and metamyelocytes peaked by day 7, as neutrophil numbers continued to increase. Based on these initial experiments, RNA was extracted from 32Dcl3G cells grown in the presence of G-CSF for 5 days and 7 days. The RDA analysis compared the pooled RNA samples from the G-CSF-treated cells against pooled RNA samples from cells grown in the absence of G-CSF (but in the presence of IL-3) for an equal number of days. This procedure yielded multiple clones, which were confirmed to be differentially expressed in G-CSF-treated cells by Northern blot analysis of the pooled RNA samples (data not shown). BLAST queries of the deduced amino acid sequences for two of the clones isolated (designated G8 and G11), indicated that both clones had significant homology to different portions of the published sequence for the hLRG protein (Fig. 2 ). The 121-bp G8 clone showed significant homology over a 40 amino acid stretch with the published sequence for the 312 amino acid hLRG protein (F11 to N51). The deduced amino acid sequence of the 436-bp G11 clone was found to be homologous to a more distal stretch of 145 amino acids (D87 to D231) in hLRG. Subsequent sequence analyses indicated that 20% of the differentially expressed clones were LRG. Differentially expressed RDA clones corresponding to MPO, migration-inhibitory factor-related protein 14, and ras-related nuclear protein were also identified.



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  		Figure 1. Time course of morphologic changes associated with G-CSF-induced neutrophilic differentiation of 32Dcl3G cells. Cells were grown in the presence of G-CSF (10 ng/mL) for the indicated times, cytospun, then stained with Wright-Giemsa solution, and examined by light microscopy. At each time point, a differential cell count on 100 cells was performed. Data are the averages from three independent experiments.

 


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  		Figure 2. Alignment of deduced amino acid sequences for hLRG and mLRG, indicating significant homology. The deduced amino acid sequences from the mLRG and hLRG cDNAs are shown and their alignment, as determined by ClustalW (version 1.81). Black and gray shading indicate identical and homologous amino acids, respectively. Sequences corresponding to the original G8 (•—•) and G11 ({blacklozenge}–{blacklozenge}) clones identified by RDA are indicated. Predicted signal peptides are shown by the double underlines. The predicted mature peptide encoded by the hLRG cDNA is identical to the previously published sequence for hLRG that was purified from serum.

 
Identification, sequencing, and analysis of mLRG and hLRG cDNAs
As RDA does not yield full-length cDNA clones, BLAST queries of the murine and human databases were performed to identify EST clones with homology to the murine RDA clones. Three murine IMAGE clones with homology to the G8 and G11 RDA clones were purchased and characterized. Sequence analysis of the 3' and 5' ends of each of the clones and size determination of the cDNA inserts by restriction analysis suggested that one clone (IMAGE clone ID: 597689) most likely contained a full-length cDNA for mLRG. Both strands of the 1310-bp cDNA insert of this clone were sequenced, and sequence analysis identified a Kozak consensus sequence, a 342 amino acid ORF, a 276-bp 3' untranslated region (UTR), and a poly-A tail (GenBank accession: AF403429). Except for 3 bp upstream from the putative translation start site, no 5' UTR was present. The deduced amino acid sequence of the ORF shares 66% identity and 76% homology with the hLRG protein sequence (Fig. 2) . SignalP V2.0 predicts a signal peptide at amino acids 1–32 (signal peptide probability=0.967, signal anchor probability=0.000, and a maximum cleavage site probability at residue 33 of 0.69). Analysis of the sequence by TargetP V1.0 suggests that mLRG is targeted to the secretory pathway.

BLAST searches of the human EST database, along with analysis of the UniGene cluster (Hs10844), identified several EST clones with homology to the LRG protein sequence. Alignment of these ESTs indicated the existence of two possible classes of LRG transcripts (designated classes I and II) differing only in the size of the 3' UTR. IMAGE clones with the largest cDNA inserts from each class were sequenced. The class I clone (IMAGE clone ID: 2426875), which was 1261-bp in length, contained a very small 5' UTR, an ORF that encoded a putative signal peptide, followed by the entire amino acid sequence of hLRG (as determined by Takahashi et al. [2 ]), and a 191-bp 3' UTR that contained an Alu repetitive element extending from bp 1133 to 1192.

The class II clone (IMAGE clone ID: 2403704) was identical to the class I clone except for a much longer 726-bp 3' UTR (GenBank accession: AF403428). Analysis of the deduced amino acid sequence of the hLRG coding region predicted a signal peptide at amino acids 1–35 (signal peptide probability=0.992, signal anchor probability=0.008, and maximum cleavage site probability at residue 36 of 0.597). Similar to mLRG, TargetP V1.0 suggests that hLRG is a secreted protein.

On the basis of these results, we have identified one mLRG and two hLRG cDNA clones. The mLRG cDNA is 1310-bp in length, and the two hLRG cDNAs are 1261-bp and 1801-bp in length.

Analysis of LRG expression
Induction of expression of LRG during G-CSF-induced granulocytic differentiation of 32Dcl3G cells was analyzed by Northern blotting (Fig. 3 A ). An approximate 1.6-kb transcript could be detected as early as 16 h after the addition of G-CSF to the culture medium. Expression of this transcript steadily increased thereafter, with an 80-fold increase at 5 days.



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  		Figure 3. LRG expression is induced during neutrophilic differentiation of 32Dcl3G cells and precedes expression of the secondary and tertiary granule enzymes LF and GEL but follows MPO. 32Dcl3G cells were grown in the presence of G-CSF (10 ng/ml) for the indicated times and RNA-extracted. (A) Northern blot analysis using a probe specific for mLRG. For each time point, 10 µg total RNA was loaded. RNA molecular weight markers are shown to the right. In the lower panel, the blot in the upper panel was stripped and reprobed with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific probe to control for RNA loading. (B) RT-PCR analysis of the expression of MPO, LF, GEL, and GAPDH. The PCR products were size-fractionated on agarose/TAE gels (1% w/v) and visualized with ethidium bromide staining.

 
We next investigated the time course for LRG expression relative to other genes known to be induced and/or up-regulated during neutrophilic granulocyte differentiation (Fig. 3B) . MPO was constitutively expressed in 32Dcl3G cells, whereas no transcripts for LF or GEL could be detected in untreated 32Dcl3G cells. G-CSF treatment of 32Dcl3G cells resulted in up-regulation of MPO expression and also in the induction of expression of LF and GEL at 3 days and 5 days, respectively. On the basis of these findings, we conclude that expression of LRG follows the expression of MPO in 32Dcl3G cells but precedes the expression of LF and GEL. These results suggest that the expression of LRG is a relatively early event in neutrophilic granulocyte differentiation.

We also examined the expression of LRG in two human myeloid cell lines that differentiate along the neutrophilic granulocyte pathway. Treatment of the human promyelocytic leukemia cell line HL-60 with DMSO, which induces granulocytic differentiation [9 ], resulted in a twofold increase in the level of the 1.8-kb LRG transcript by day 7 (Fig. 4 A ). Neutrophilic granulocyte differentiation of the HL-60 cells was confirmed morphologically by Wright-Giemsa staining and the NBT assay. In contrast, treatment with PMA, which induces monocytic differentiation of HL-60 cells [28 ], resulted in a decrease in LRG expression. The low level of basal expression of LRG detected in untreated HL-60 cells may reflect a more mature phenotype of HL-60 cells, as compared with 32Dcl3G cells. The observation that DMSO-induced granulocytic differentiation of HL-60 cells is associated with up-regulation of LRG expression demonstrates that this effect is not unique to G-CSF.



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  		Figure 4. LRG expression is up-regulated during neutrophilic differentiation of HL-60 and MPD cells but down-regulated during monocytic differentiation. (A) HL-60 cells treated with PMA (5 nM) or DMSO (1.25% v/v) to induce monocytic or neutrophilic differentiation, respectively. (B) MPD cells treated with G-CSF (2.8 ng/ml) to induce neutrophilic differentiation. At each time point, RNA was extracted for analysis by Northern blotting using a probe for hLRG. For each time point, 10 µg total RNA was loaded. Following hybridization with the LRG probe, each blot was stripped and reprobed with an 18S rRNA-specific probe (lower panels). Molecular weight markers are shown to the right. Treatment of HL-60 cells was carried out for 2 days, at which time the cells became adherent consistent with a monocyte/macrophage phenotype.

 
We also observed up-regulation of LRG expression during neutrophilic granulocyte differentiation in the human MPD cell line, which differentiates in response to G-CSF, and expression of proteins belongs to all three classes of neutrophilic granules [10 ]. Similar to HL-60 cells, LRG expression was also detected in untreated MPD cells. G-CSF treatment of MPD cells resulted in an increase in expression of the 1.8-kb LRG transcript, which could be detected as early as 24 h after the addition of rhG-CSF to the culture medium (Fig. 4B) . LRG expression in MPD cells increased steadily thereafter, with an approximate threefold increase by day 14. In HL-60 and MPD cells, expression of only the class II LRG transcript was detected.

We next investigated the expression of LRG in primary cells from healthy volunteer donors. LRG transcripts were detected in the neutrophil fraction and in the progenitor-rich mononuclear cell fraction of BM (Fig. 5 ). In contrast, LRG expression could only be detected in the neutrophil fraction from PB and not in the mononuclear cell fraction. Similar to the results obtained with cell lines, only class II transcripts for hLRG could be detected in BM and PB.



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  		Figure 5. LRG is expressed in neutrophils and bone marrow mononuclear cells but not in peripheral blood mononuclear cells. Mononuclear cells (M) and neutrophils (N) were isolated from BM and PB samples from healthy donors as described in Materials and Methods. Total RNA (10 µg) from each fraction was examined for LRG expression by Northern blot analysis. A representative blot is shown. Similar results were obtained using four different donors. In the lower panel, the blot in the upper panel was stripped and reprobed with an 18S rRNA-specific probe.

 
We also examined the tissue distribution for expression of LRG. Northern blot analysis of mRNA samples extracted from adult mouse tissues demonstrated a high level of LRG expression in liver with a much lower level in heart and minimally detectable expression in spleen and lung. No LRG transcripts were detected in brain, skeletal muscle, kidney, or testis (data not shown).

Chromosomal localization and genomic structure of mLRG and hLRG
A BLAST query of the public Celera Inc. human genome sequence database using the sequence of the human class I clone indicated a 100% match to segment GA xx2HTBKPS83T:1000001:1500000, which localizes to chromosome 19. Electronic PCR of the 6000-bp surrounding the hLRG genomic locus identified two sequence tag sites, stSG44969 and sts-T71373, in the 3' UTR of the hLRG locus. These sequence tag sites belong to the UniGene cluster Hs10844 and have been mapped to 19p13.3 by radiation hybrid mapping [29 ]. Using BLAST2, alignment of the cDNA sequences for hLRG with the genomic sequence for hLRG indicated that the hLRG gene is composed of two exons. Exon 1 is 56-bp in length and contains 25-bp of the 5' UTR and the first 32-bp of the 1044-bp ORF. Exon 2 is 1737-bp in length, and contains the remainder of the ORF, including the stop codon and the entire 722-bp 3' UTR. The intervening intron is 1030-bp in length and contains consensus splice donor and acceptor sites. Analysis of the genomic sequence upstream from the hLRG coding region indicated a putative transcription start site approximately 30 bp upstream of the ATG. Comparison of the putative promoter regions identified for the hLRG and mLRG genes revealed the presence of clusters of high homology and approximately 60% overall identity (Fig. 6 A ).



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  		Figure 6. Comparison of putative LRG promoter regions of the hLRG and mLRG genes. (A) Pustell DNA dot matrix comparison of the 5' flanking regions. (B) Alignment of human and murine sequences with the putative TATA boxes and the ATG start sites underlined. Boxes denote sequences corresponding to putative binding sites for transcription factors.

 
The genomic sequence 500 bp upstream and 100 bp downstream of the putative transcription start site was examined for consensus sequences for DNA binding sites for all transcription factors in the TRANSFAC database [23 ]. This analysis identified putative binding sites for C/EBP, PU.1, MZF1, and STAT (Fig. 6B) .

A BLAST query of the mouse-specific genome database using the mLRG cDNA sequence identified a BAC clone (GenBank accession: AC026385), which mapped to mouse chromosome 11 and contained a portion of the mLRG genomic sequence, including the putative promoter region, the entire 5' UTR, and a portion of the coding sequence. Alignment of the mLRG cDNA to this BAC indicated the presence of at least two exons in the mLRG genomic locus. Similar to the hLRG gene, exon 1 of the mLRG gene is small at 44 bp in length and contains the first 26 bp of the 1029-bp ORF preceded by a 5' UTR of approximately 18 bp. The intron is 916 bp in length and contains consensus splice-donor and splice-acceptor sites. The sequence of the second exon is incomplete, as the BAC sequence terminates before the end of this exon. Genomic sequence data containing the remainder of the mLRG gene are not currently available. However, the available structure for the mLRG gene is very similar to that of the hLRG gene. Analysis of the genomic sequence upstream of the identified mLRG coding region predicted a transcription start site approximately 30 bp upstream of the ATG. Sequence analysis of the putative mLRG promoter region indicated the presence of putative binding sites for C/EBP, MZF1, and STAT (Fig. 6B) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The leucine-rich repeat family is a diverse family of proteins, which have been shown to be involved in protein-protein interactions, signal transduction, and cell adhesion and development [3 ]. The function of the founding member of this family, LRG, has remained unknown. Here, we report expression of LRG in primary human neutrophils and the induction and/or up-regulation of its expression during neutrophilic granulocyte differentiation of human and murine cell lines.

Using cDNA RDA, we identified LRG as being differentially expressed during G-CSF-induced granulocytic differentiation of murine 32Dcl3G cells. BLAST queries using the RDA clones enabled us to obtain full-length cDNA clones for mLRG and hLRG from the EST clone banks. Two classes of hLRG cDNA were identified, which differed only in the length of their 3' UTRs. Comparison of the sequences of the two human mRNA classes with the LRG genomic sequence indicates that the difference is not a result of alternative splicing but rather because of different polyadenylylation sites. The polyadenylylation site of the class II clone has a canonical AAUAAA signal sequence, but the shorter class I clone does not. It is possible that the class I clone is not a true mRNA species but rather an artificial species produced by the cloning method. As the sequence surrounding the putative polyadenylylation site in the class I clone is rich in adenosine bases, mispriming the oligo-dT primer during cDNA synthesis could have given rise to the class I clones. In any event, the only class of LRG transcript detected in cell lines and in primary human cells from PB and BM was the class II transcript.

Expression of LRG in 32Dcl3G cells could be detected as early as 16 h following G-CSF treatment, nearly 4 days prior to the appearance of cells with the morphological features of neutrophils. The time course for induction of LRG expression in relation to other genes known to be up-regulated during granulopoiesis provided additional evidence that LRG expression is induced at an early time point during neutrophilic granulocyte differentiation. LRG expression was found to occur subsequent to the expression of myeloperoxidase, a primary/azurophilic granule protein, but prior to the expression of lactoferrin and gelatinase, which are contained within the secondary and tertiary granules, respectively. In addition, LRG transcripts were detected in the granulocytic and mononuclear cell fractions of bone marrow and in peripheral blood granulocytes but not monocytes or lymphocytes. Collectively, these data suggest that LRG expression during hematopoiesis is specific for differentiation along the neutrophilic granulocyte lineage and that its expression is induced early during this process.

Of interest is the identification of consensus binding sites for myeloid-specific transcription factors in the putative promoter regions of the hLRG and mLRG genes. The C/EBP family of transcription factors has been shown to be involved in early myeloid differentiation [30 , 31 ]. C/EBP{alpha}-null mice exhibit a blockade in granulocyte development at the myeloblast stage, and C/EBP{varepsilon}-null mice were found to have an increase in the numbers of immature myeloid cells in their bone marrow and produced hyposegmented neutrophils that were functionally deficient [32 , 33 ]. An absolute requirement for PU.1 during granulopoiesis has been demonstrated in vitro and in vivo [34 , 35 ]. Two PU.1-deficient mouse strains have been generated. In one case, no viable PU.1-/- progeny were produced (embryos died by day 18), whereas in the other case, newborn pups died of severe septicemia after 48 h. Both PU.1-null mouse strains were found to be deficient in multiple hematopoietic lineages, including monocytes, neutrophils, B cells, and T cells [36 , 37 ]. Notably, binding sites for PU.1 and C/EBP{alpha} have also been identified in the promoter region for other myeloid-specific genes, including myeloperoxidase, neutrophil elastase, and the G-CSF receptor [38 39 40 ].

Consensus binding sites for MZF-1 and STAT were also identified in the putative promoters of the hLRG and mLRG genes. G-CSF-stimulated granulocyte-colony formation has been reported to be inhibited in vitro by antisense oligonucleotides to MZF-1, and STATs have been shown to mediate signaling via many cytokine receptors, including the G-CSF receptor [41 , 42 ].

Results from the TargetP algorithm suggest that LRG is processed along the secretory pathway, consistent with its initial purification from plasma. The SignalP V2.0 algorithm predicts a signal peptide of 35 residues for the human protein and 32 residues for the mouse protein. Although a signal peptide of 35 (or 32) amino acids is unusually long, the predicted cleavage site would result in a protein that is 100% homologous to the primary sequence previously reported for hLRG. The absence of a signal-anchor region, as indicated by the SignalP V2.0 analysis, further supports the notion that LRG is a secreted protein. It is possible that LRG is secreted directly or that it is stored in intracytoplasmic granules prior to secretion, similar to other secreted neutrophil granule proteins such as myeloperoxidase, lactoferrin, and gelatinase. The lack of any currently available antibodies to LRG has hindered the determination of its cellular localization. Future studies using antibody generated against purified LRG as well as epitope-tagged LRG and commercially available antibodies recognizing the tag, which are currently underway in our laboratory, should help to elucidate the cellular localization of LRG and its physiologic role in neutrophilic granulocytes.


   ACKNOWLEDGEMENTS
 
This work was supported by grants CA75226, CA82859, and CA16058 from the National Cancer Institute. We thank Drs. Jas Lang, Giovanni Rovera, Michael Baumann, Cassandra Paul, and Harvey Lodish for generously providing the cell lines used in our studies.

Received December 20, 2001; revised March 21, 2002; accepted May 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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