Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3319-3323, May 1988 Biochemistry Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b CHARLES A. PARKOS*, MARY C. DINAUERt, LESLIE E. WALKER*, RODGER A. ALLEN*, ALGIRDAS J. JESAITIS*, AND STUART H. ORKINtt *Department of Immunology, Research Institute of the Scripps Clinic, La Jolla, CA 92037; tDivision of Hematology-Oncology, Children's Hospital, and Dana-Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Boston, MA 02115; and tHoward Hughes Medical Institute, Children's Hospital, Boston, MA 02115 Communicated by Harvey F. Lodish, January 14, 1988 ABSTRACT Cytochrome b comprising 91-kDa and 22- Cytochrome b purified from neutrophil membranes ap- kDa subunits is a critical component of the membrane-bound pears to be a heterodimer of a glycosylated 91-kDa heavy oxidase of phagocytes that generates superoxide. This impor- chain and a nonglycosylated 22-kDa light chain (10-12). The tant microbicidal system is impaired in inherited disorders 91-kDa subunit is encoded by a gene designated CGD, known as chronic granulomatous disease (CGD). Previously we residing at chromosomal position Xp2l, which originally was determined the sequence of the larger subunit from the cDNA identified on the basis of genetic linkage without reference to of the CGD gene, the X chromosome locus affected in "X- a specific protein product (8). Antisera generated to either a linked" CGD. To complete the primary structure of the synthetic peptide predicted from the cDNA or to a fusion cytochrome b and to assess expression of the smaller subunit, protein produced in E. coli recognized the 91-kDa component we isolated cDNA clones for the 22-kDa polypeptide by of purified cytochrome b (9). Furthermore, direct N-terminal immunoscreening and confirmed their authenticity by direct amino acid sequencing confirmed the nature of the predicted N-terminal protein sequencing. Although the deduced amino protein (13). Since the sequence of the 91-kDa component acid sequence of the 22-kDa subunit is not overtly similar to demonstrated no significant similarity to known cytochromes at the primary amino acid sequence level (8), the relative other known cytochromes, we observed a 31-amino acid stretch function of the two subunits, the location of the heme pros- of 39% identity with polypeptide I of mitochondrial cyto- thetic group in the heterodimer, and the nature of the associ- chrome c oxidase centered on a potential heme-coordinating ation between the subunits remain unknown. Further under- histidine. Similarities in the hydropathy profiles and spacing of standing of the structure of the cytochrome b heterodimer histidines of the 22-kDa protein and myoglobin suggest struc- requires characterization of the smaller subunit. tural motifs in common with other heme-containing proteins Here we report the isolation and nucleotide sequence of that are not readily revealed by primary amino acid sequences. cDNA clones encoding the 22-kDa subunit,§ and confirma- Although RNA for the larger subunit has been found only in tion of the deduced N-terminal amino acids by direct se- cells ofthe phagocytic lineage, stable RNA encoding the 22-kDa quencing of the 22-kDa subunit. Comparison of the primary subunit was observed in all cell types. However, the stable structure of the two subunits with known cytochromes 22-kDa protein was detected only in phagocytic cells that were suggests that the cytochrome b is, indeed, an atypical expressing the larger subunit RNA. This observation suggests cytochrome species. In contrast to the mRNA of the CGD that the large subunit may play a role in regulating the gene encoding the 91-kDa subunit, which is highly regulated assembly of the heterodimeric cytochrome b. in a lineage-specific manner (8), the transcript for the 22-kDa subunit appears to be expressed constitutively in a variety of Phagocytic cells (neutrophils, macrophages, and eosinophils) cell types, even though the polypeptide itself appears to be are central to the host defense against invading microbes (1, present in stable form only within phagocytic cells. 2). Upon stimulation with a variety of particulate or soluble agents, a latent NADPH-oxidase system is activated and METHODS produces large quantities of superoxide (3-5). This radical is converted to toxic oxygen derivatives important for micro- Isolation and Sequencing of cDNA Clones. Bacteriophage bicidal activity and inflammatory tissue injury (1, 2, 6). The cDNA libraries in the A phage vectors Agtll and AgtlO, protein components of the oxidase have been incompletely constructed with mRNA derived from human promyeleocy- defined, but evidence suggests that it is a nonmitochondrial, tic leukemia HL60 cells, have been described (8). Agtll plasma membrane-bound electron transport system that recombinants were screened essentially as described (14) by includes a b-type cytochrome with an unusually low redox using the IgG fraction of a polyclonal antiserum directed to potential and probably a flavoprotein (4, 5, 7). Compelling the 22-kDa polypeptide (10). To obtain a full-length cDNA evidence for the functional importance of the cytochrome b clone, the EcoRI insert of clone 12 (see Results) was is provided by studies ofthe genetic disorder X chromosome- radiolabeled by random priming (15) and used to screen the linked chronic granulomatous disease (X-CGD) (7), which AgtlO library by plaque hybridization (16, 17). Nucleotide results from mutations in a gene now known to encode one sequences subcloned in phage M13 derivatives were deter- component of the cytochrome (refs. 8 and 9 and see below). mined by the dideoxynucleotide chain-termination method This disorder is characterized by the inability of phagocytes (18, 19). to produce superoxide (7). Consequently, affected individu- Purification of Neutrophil Cytochrome b, Amino Acid Se- als lack an essential host microbicidal system and develop quencing, and Immunoblot Analysis. Neutrophil cytochrome recurrent, severe bacterial and fungal infections. Abbreviations: CGD, chronic granulomatous disease; X-CGD, X chromosome-linked CGD. The publication costs of this article were defrayed in part by page charge §The sequence reported in this paper is being deposited in the payment. This article must therefore be hereby marked "advertisement" EMBL/GenBank database (Intelligenetics, Mountain View, CA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03774). 3319 3320 Biochemistry: Parkos et al. Proc. Natl. Acad. Sci. USA 85 (1988) b was purified by the procedure of Parkos et al. (10). Final with FAST-P. Hydropathy analyses and plots were obtained purification for protein sequencing was achieved by one- from application of CHOUFASMAN and PEPPLOT dimensional NaDodSO4/PAGE and electroelution onto a (UWGCG) programs. These programs apply the Kyte and poly(vinylidene difluoride) membrane essentially as de- Doolittle method (24) to calculate a hydrophobicity index for scribed by Matsudaira (20). Sequence analysis (20) of the each amino acid residue averaged over a window of nine 22-kDa band was performed with an Applied Biosystems residues. (Foster City, CA) model 470 sequenator equipped with on-line phenylthiohydantoin analysis using the program 03RPITH and reverse-phase HPLC on a Brownlee C18 RESULTS column. Isolation of cDNA for the 22-kDa Subunit of Neutrophil Electrophoretic immunoblots were performed as described Cytochrome b. Polyclonal antiserum directed to the 22-kDa (9-11). subunit ofthe cytochrome b purified from human neutrophils RNA Blot Hybridization and S1 Nuclease Protection Anal- (10) was used to screen a Agtll expression vector library (8) ysis. Total cellular RNA was isolated from cultured cells as prepared from dimethylformamide-treated HL60 cells. Initial described (7) and subjected to blot analysis (17). For S1 screening of 700,000 recombinants identified 26 positive nuclease protection assays, the cDNA insert from clone 17 clones. All except 1 clone displayed high-stringency cross- was digested with BstEII, 5'-end-labeled with 'y-ATP and hybridization. A 680-base-pair EcoRI fragment of clone 12 polynucleotide kinase, and hybridized to total cellular RNA was used to isolate a full-length cDNA (clone 17) from the samples under conditions as described (21). RNADNA AgtlO library. hybrids were digested with 750 units of S1 nuclease per ml Fig. 1 displays the nucleotide sequence of the putative and electrophoresed under denaturing conditions in urea/ cDNA for the 22-kDa subunit and the predicted amino acid acrylamide gels. sequence. The sequence of 680 nucleotides [excluding the Expression of 22-kDa cDNA in COS Cells. For transient poly(A) tract] reveals a potential open reading frame extend- expression of the cDNA encoding the 22-kDa subunit, the ing from the ATG at nucleotide positions 29-31 to a termi- EcoRI sites at the ends ofclone 17 were converted to HindIII nation codon (TGA) at 614-616. A consensus polyadenyly- sites with synthetic linkers, and the fragment was recloned in lation signal (25) (AATAAA) is present at positions 659-664. pSV-HdIII (22). Monkey kidney COS cells were transfected The predicted primary translation product of 195 amino acids by the procedure of Chen and Okayama (23) and harvested has an estimated Mr of 20,961. The deduced primary amino for immunoblots 48 hr after removal ofthe calcium phosphate acid sequence is notable for its relatively high proline content precipitate from the cells. (10%). Of the 20 predicted proline residues, 17 lie within the Computer Analyses. Computer analysis of the primary C-terminal 63 amino acids. The directly determined N- structure was performed on a Digital Equipment Corporation terminal sequence of 25 amino acids obtained from the VAX computer utilizing the software available from the Uni- purified 22-kDa polypeptide (Fig. 2) established that the versity of Wisconsin Genetics Computer Group (UWGCG) cDNA isolated by antibody screening is derived from the and the Protein Identification Resource of the Division of mRNA that encodes the 22-kDa subunit.