Potential Asparagine-linked Glycosylation Sites Research Articles

26P Structurally constrained tumor-specific epitope within the largest extracellular domain of sodium-dependent phosphate transporter NaPi2b

The search for tumor-specific targets among membrane proteins is important for the development for new effective anticancer drugs because membrane proteins represent 60% of drug targets. Transmembrane proteins in cancer cells exhibit an altered pattern of post-translational modifications and generally contain a large extracellular domains with multiple glycosylation and sulfhydryl sites with unknown function. We analyze the features of membrane protein which are required for recognition by monoclonal antibodies using the sodium-dependent phosphate transporter NaPi2b, containing a large extracellular domain 4 (EMD4, 250-360 a.a.) with 4 cysteines and 6 potential asparagine-linked glycosylation sites which can potentially contribute to foldability of conformationally exposed epitope corresponding to this domain.

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity.

Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug.

Analysis of the Fusion Protein Gene of Newcastle Disease Viruses Isolated in Japan

The complete nucleotide sequences of the fusion (F) protein gene of Newcastle disease viruses (NDV) isolated in Japan from 1930 to 2007 (45 strains total) were determined and genetically analyzed. In the deduced amino acid sequences of fusion protein, the 5 potential asparagine-linked glycosylation sites and 10 cysteine residues were all conserved in the NDV examined in this study. The major epitopes involved in virus neutralization are conserved in most of the NDV strains isolated in Japan except a few strains. By virus neutralization test, no major antigenic differences were observed among representative strains of each genotype in Japan. All chickens vaccinated with the B1 strain survived without clinical signs after challenge with 2 NDV strains isolated in Japan (velogenic strains, JP/Ibaraki/2000 and JP/Kagoshima/91), which possess amino acids substitutions involved in virus neutralization in the F protein gene.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10–30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be ∼4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL ∼4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by ∼7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11–15 IU mg −1) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

CDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5′-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, comfirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Membrane organization of bluetongue virus nonstructural glycoprotein NS3.

The smallest RNA segment (S10) of bluetongue virus (an orbivirus, family Reoviridae) encodes two closely related nonstructural proteins, the 229-amino-acid (aa) NS3 and the 216-aa NS3A. The proteins are found in glycosylated and nonglycosylated forms in infected cells (X. Wu, H. Iwata, S.-Y. Chen, R. W. Compans and P. Roy J. Virol. 66:7104-7112, 1992). The NS3/NS3A proteins have two hydrophobic domains (aa 118 to 141 and 162 to 182) and two potential asparagine-linked glycosylation sites (aa 63 and 150), one of which is located between the hydrophobic domains. To determine whether these features were used in the mature protein forms, we generated a series of mutants of the S10 gene and expressed them by using the vaccinia virus T7 polymerase transient-expression system. Our data indicate that both hydrophobic domains of NS3 span the cell membrane and that only the site at aa 150 is responsible for N-linked glycosylation of the NS3 proteins.

Isolation and Characterization of an Invertase and Its Repressor Genes fromSchizosaccharomyces pombe

PCR was used to isolate an invertase homolog gene from the fission yeastSchizosaccharomyces pombe.The clonedinv1+gene encodes a protein of 581 amino acids with 16 potential asparagine-linked glycosylation sites, and has 39% and 38% identity to theSchwanniomyces occidentalisandSaccharomyces cerevisiae SUC2invertases. When theinv1+gene was disrupted,S. pombestrains lacked detectable invertase activity. This result showed that theinv1+gene encodes only one active invertase inS. pombecells. The transcription ofinv1+is repressed in the presence of glucose. The transcription ofinv1+was not affected incyr1Δ strain which lacks adenylate cyclase activity, unlike transcription ofS. pombe fbp1+gene. We have identified anS. pombegene (scr1+) that encodes a homolog of theAspergillus nidulansCREA which is required for glucose repression of the glyconeogenic pathway. Although the deletion ofscr1+did not influence the transcription offbp1+gene, glucose repression of theinv1+gene was severely affected. These results showed that glucose repression ofinv1+gene is dependent onscr1+gene, andS. pombecAMP signalling pathway may not be essential for glucose repression ofinv1+gene.

The nucleotide and predicted amino acid sequence of the fusion protein of recent isolates of canine distemper virus in Japan.

Analysis of the molecular properties of fusion (F) proteins of field isolates of canine distemper virus (CDV) by immunoprecipitation analysis revealed an identical molecular mass of F protein of 3 field isolates as well as the Onderstepoort laboratory strain. Sequencing showed that the F gene of a field isolate (the Yanaka strain) shared 90.1% and 95.7% identities with the Onderstepoort strain at nucleotide and amino acid levels, respectively. All of the 13 cysteine residues and 4 potential asparagine-linked glycosylation sites were completely conserved amongst these strains. These results indicate that the F proteins is much less heterogeneous than that observed in the hemagglutinin proteins of CDV.

Cloning and expression of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2

The human liver cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT) was isolated from a lambda gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There were three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to obtain UDPGTh2 protein encoded from cloned human liver UDP-glucuronosyltransferase cDNA, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. The presence of a transferase with Mr approximately 52,000 in transfected cells cultured in the presence of [(35)S]methionine was shown by immunocomplexed products with goat antimouse transferase IgG and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The expressed UDPGT was a glycoprotein as indicated by electrophoretic mobility shift in Mr approximately 3,000-4,000 when expressed in the presence of tunicamycin. The extent of glycosylation was difficult to assess, although one could assume that glycosyl structures incorporated at the level of endoplasmic reticulum were always the core oligosaccharides. Thus, it is likely that at least two moieties inserted can account for the shift of Mr approximately 3,000-4,000. This study demonstrates the cDNA and deduced amino acid sequence of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.

Molecular Cloning, Primary Structure, and Properties of a New Glycoamidase from the Fungus Aspergillus tubigensis

A new glycoamidase, peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked alpha1-->6 or alpha1-->3 fucose, under conditions not favorable with existing glycoamidases.

Molecular Cloning and Expression of a Bovine α(1,3)-Fucosyltransferase Gene Homologous to a Putative Ancestor Gene of the Human FUT3-FUT5-FUT6 Cluster

Only one bovine gene, corresponding to the human cluster of genes FUT3-FUT5-FUT6, was found by Southern blot analysis. The cognate bovine alpha(1,3)-fucosyltransferase shares 67.3, 69.0, and 69.3% amino acid sequence identities with human FUC-T3, FUC-T5, and FUC-T6 enzymes, respectively. As revealed by protein sequence alignment, potential sites for asparagine-linked glycosylation and conserved cysteines, the bovine enzyme is an intermediate between FUC-T3, FUC-T5, and FUC-T6 human enzymes. Transfected into COS-7 cells, the bovine gene induced the synthesis of an alpha(1, 3)-fucosyltransferase enzyme with type 2 substrate acceptor pattern specificity and induced expression of fucosylated type 2 epitopes (Lex and sialyl-Lex), but not of type 1 structures (Lea or sialyl-Lea), suggesting that it has an acceptor specificity similar to the human plasma FUC-T6. However, no enzyme activity was detected in bovine plasma. Gene transcripts are detected on tissues such as bovine liver, kidney, lung, and brain. The type 2 sialyl-Lex epitope was found in renal macula densa and biliary ducts, and Lex and Ley epitopes were detected on the brush border of epithelial cells of small and large intestine, suggesting a tissue distribution closer to human FUC-T3, but fucosylated type 1 structures (Lea, Leb, or sialyl-Lea) were not detected at all in any bovine tissue. Analysis of genetic distances on a combined phylogenetic tree of fucosyltransferase genes suggests that the bovine gene is the orthologous homologue of the ancestor of human genes constituting the present FUT3-FUT5-FUT6 cluster.

Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs.

We isolated three strains of canine distemper virus (CDV)--the Ueno, Hamamatsu, and Yanaka strains--from dogs in Japan and analysed the molecular properties of their haemagglutinin (H) proteins. Immunoprecipitation of all three strains with a monoclonal antibody revealed H proteins with molecular masses of 84 kDa, which differs from the molecular mass (78 kDa) of the H protein of the Onderstepoort vaccine strain. However, after tunicamycin treatment immunoprecipitation identified H proteins of identical molecular mass (68 kDa) for all three field isolates and the vaccine strain. Sequence analysis showed nine potential sites for asparagine-linked glycosylation in the H proteins of the new isolates, in contrast to four in the H protein of the Onderstepoort strain. Thus, variation in glycosylation of the H proteins of the isolates and the vaccine strain may cause differences in antigenicity of the viruses. Sequences of the H genes showed that the new Japanese isolates have 99% identity with each other, 95% with other European and American isolates (from seals, a German dog, a ferret and large felids) and 90% with the vaccine strain. Phylogenetically, the new Japanese isolates form one cluster which is separate from recent European or American isolates, all of which are distinct from vaccine strains.

Cloning and sequence analysis of the human MG160, a fibroblast growth factor and E-selectin binding membrane sialoglycoprotein of the Golgi apparatus.

The amino acid sequence of MG160, a membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, is more than 90% identical with CFR, a fibroblast growth factor (FGF) binding protein of chicken membranes, and with ESL-1, a ligand for E-selectin of plasma membranes of myeloid cells; furthermore, MG160, isolated by immunoaffinity chromatography from rat brain membranes, binds to basic FGF. The gene for MG160 has been assigned to human chromosome 16q22-23. To characterize this protein further in the human, its cDNA was cloned and sequenced. The protein has a large luminal domain composed of an initial proline-glutamine-rich segment, encoded by an uninterrupted exonic sequence of several CAG-CAA repeats. Expansion of CAG repeats has been implicated in the etiology of several neurodegenerative diseases. The proline-glutamine-rich segment is followed by 16 cysteine-rich repeats that contain five potential asparagine-linked glycosylation sites, which are conserved in the human, rat, mouse, and chicken. The large intralumenal domain of the protein is followed by a single transmembrane domain and a 13-amino-acid cytoplasmic carboxy-terminal tail, which is identical to that in the chicken, rat, and mouse. The overall amino acid identifies between MG160, CFR, and ESL-1 range from 88% to 95%. In several human fetal and adult tissues, three mRNA transcripts for MG160 of 10 kb, 5 kb, and 3.8 kb were identified by Northern blot analysis of poly(A)-selected mRNAs. These transcripts may represent alternatively spliced mRNAs of the protein or mRNAs encoding closely related proteins of the Golgi apparatus and/or plasma membranes.

Isolation and Characterization of a Novel cDNA Encoding a Human UDP-Glucuronosyltransferase Active on C19 Steroids

To isolate cDNA clones encoding novel UGT2B enzymes, human prostate and LNCaP cell cDNA libraries were screened using a pool of steroid-specific UGT2B cDNA probes. In approximately 10(6) recombinants, we isolated 3 cDNA clones of 2.1 kilobases that encode a novel UGT2B enzyme. UGT2B17 is 95% identical with UGT2B15 and 91% identical with UGT2B8. Primary structure analysis of UGT2B17 based on the nucleotide sequence revealed a putative amino-terminal membrane insertion signal peptide, a carboxyl-terminal membrane-spanning region, and three potential asparagine-linked glycosylation sites. UGT2B17 cloned in the pBK-CMV expression vector was transfected into HK293 cells to obtain a stable clonal cell line expressing a high level of the active 53-kDa UGT2B17 enzyme. Of the over 60 endogenous and exogenous substances tested, 25 compounds revealed reactivity. The major substrates are eugenol > 4-methylumbelliferone > dihydrotestosterone > androstane-3alpha, 17beta-diol (3alpha-diol) > testosterone > androsterone (ADT). The apparent Km values obtained with tritiated steroids in intact cells were 0.4 microM for ADT, 0.7 microM for dihydrotestosterone, 1.0 microM for 3alpha-diol, and 3.4 microM for testosterone. Southern blot analysis of reverse transcription-polymerase chain reaction products revealed expression of UGT2B17 mRNA in various tissues including the liver, kidney, testis, uterus, placenta, mammary gland, adrenal gland, skin, and prostate. UGT2B17 is the first human uridine diphosphoglucuronosyltransferase enzyme expressed in extrahepatic tissues to have a specificity for ADT as well as testosterone, dihydrotestosterone, and 3alpha-diol.

The Major Site of Photoaffinity Labeling of the γ-Aminobutyric Acid Type A Receptor by [3H]Flunitrazepam Is Histidine 102 of the α Subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).

Molecular cloning and nucleotide sequence determination of three envelope genes of classical swine fever virus Taiwan isolate p97

A strain of classical swine fever virus (CSFV) has been isolated in Taiwan. The cDNA coding for three envelope glycoproteins E1, E2 and E3 were molecularly cloned from purified viral particles using the reverse transcription-polymerase chain reaction (RT-PCR) method and sequence-specific primers. The resulting PCR products (1113 bp for E1, 699 bp for E2 and 567 bp for E3) were cloned into the SmaI site of pUC19 and then subjected to DNA sequence analysis. Data showed that nucleotide sequence of the three envelope genes shared a 82–83% homology with the corresponding genes of three other strains (Alfort, Brescia and Weybridge). However, the homology of the deduced amino acid sequence was greater than 90% among the four strains. The potential asparagine-linked glycosylation sites for E1 (5 sites), E2 (7 sites) and E3 (2 sites) were conserved. This suggests that the Taiwan p97 strain is distinct from other three strains described. The variations may have implications for future vaccine development. The sequence has been submitted to GenBank. The accession numbers are U43924 and U03290.

Genetic and antigenic properties of Dobrava virus: a unique member of the Hantavirus genus, family Bunyaviridae.

We examined the genetic and antigenic properties of Dobrava (DOB) virus, a hantavirus associated with severe haemorrhagic fever with renal syndrome in Europe. Cloning and sequence analyses revealed the DOB M segment to consist of 3644 nucleotides, with a coding capacity of 1134 amino acids in the virus complementary-sense RNA (cRNA). Seven potential asparagine-linked glycosylation sites were identified in the M segment gene product, one in the G2 and six in the G1 coding regions. The S segment is 1667 nucleotides long, and has a single ORF in the cRNA capable of encoding a protein of 428 amino acids. Phylogenetic comparisons of the M and S segments of DOB virus to those of other hantaviruses indicated that DOB virus is similar to, but clearly distinct from Hantaan (HTN) and Seoul (SEO) viruses. Certain G2-specific, but not G1-specific monoclonal antibodies to HTN virus reacted to the same titre with DOB and homologous viral antigen. Plaque-reduction neutralization tests indicated that, of the sera tested, only antisera to SEO virus were able to neutralize DOB virus to a titre greater than 1:10; however, this neutralization titre was eightfold lower than that observed with homologous SEO virus. The data reported here confirm that DOB virus is a unique species in the Hantavirus genus, family Bunyaviridae.

A Novel Carbohydrate Addition Site on the Hemagglutinin Protein of a Highly Pathogenic H7 Subtype Avian Influenza Virus

The highly pathogenic (HP) avian influenza isolate, A/Fowl/Victoria/76 (H7N7), contains two naturally occurring hemagglutinin (HA) variants. The two hemagglutinin proteins differ only in the possession of a potential asparagine-linked glycosylation site at amino acid position 188-190, which is near the proposed receptor binding region of the HA. Expanded virus plaques which possess the addition site exhibit more slowly migrating HA1 subunits and are significantly more lethal in chickens than those which lack the site. When artificial mixtures of the two variants were inoculated in birds, as few as 1 in 1000 particles containing the glycosylation site was sufficient to exhibit 100% lethality in birds. The data raise the possibility that presence of carbohydrate near the receptor site on the H7 avian influenza virus hemagglutinin may influence virulence.

Molecular characterization and differential mRNA tissue distribution of mouse apolipoprotein D.

The mouse apolipoprotein D gene was isolated from a brain cDNA library. The nucleotide sequence contains a unique reading frame coding for a protein sharing 79.5% homology with human apoD, 86.2% homology with rabbit apoD and 92.6% homology with rat apoD. The four sequences have two potential asparagine-linked glycosylation sites at residues 45 and 78, and possess the two consensus sequences of the lipocalin family which coincide with the most conserved regions in the four species studied. The distribution of apoD mRNA among mouse organs was determined by Northern blot and quantitative dot blot analysis. The highest levels of mRNA were found in the central nervous system (CNS), namely in the spinal cord, the cerebellum and the brain. Very low concentrations were detected in all the other organs tested. In some organs (spleen, kidney, intestines, heart), a second messenger of lower molecular weight was detected. Gene expression was also measured in rat tissues. As in the mouse, rat CNS was found to be by far the highest expressor of apoD mRNA, in contrast to the rabbit and human. Levels of expression in most mouse and rat organs appeared to be much lower than in the same organs of the rabbit and human. Since apoD is expressed at sites of nerve regeneration as well as apoE, our results raise the question of whether or not the two proteins play a coordinated role in the CNS.