Fabry disease is an X-linked inborn error of glycolipid metabolism caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GalA; EC 3.2.1.22). In order to obtain large quantities of this human enzyme for physical characterization and for the development of new approaches for enzyme therapy, we constructed derivatives of the Autographa californica nuclear polyhedrosis virus that produce the human enzyme. The recombinant GalA (re-GalA) is produced at high levels, and is active with both the artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, and the natural in vivo substrate, trihexosylceramide. The purified re-GalA is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. Mass spectral analysis of total monosaccharides released by hydrazinolysis indicates that it contains fucose, galactose, mannose and N-acetylglucosamine. Amino-acid sequence analysis of six proteolytic peptides corresponded to sequences predicted by the cDNA. The molecular masses of the purified enzyme, estimated by electrospray mass spectroscopy and laser desorption time-of-flight analysis are 46.85 and 46.62 kDa, respectively, approx. 10% greater than the polypeptide portion predicted by the cDNA. The recombinant enzyme retains significant catalytic activity after modification with poly(ethylene glycol), a treatment which decreases the immunogenicity and increases the circulation life of many proteins used therapeutically.

“Neurotrophic factors” were proposed to exist following the observation, now decades old, that neurons die during normal development. This process of neuronal death, known as programmed or naturally occurring neuronal death, is thought to somehow be involved in the proper wiring of the nervous system. A variety of studies revealed that the amount of neuronal death observed was proportional to the amount of target innervated, leading to the notion that neurons compete for limiting amounts of target-derived neurotrophic activity (Hamburger and Levi-Montalcini 1949; Oppenheim 1981). The ability to assay for neurotrophic activity in vitro allowed the isolation of neurotrophic factors, the first of which was nerve growth factor (NGF) (Levi-Montalcini 1987). The subsequent purification and molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that it shared about 50% amino acid identity with NGF (Leibrock et al. 1989) and inspired homology-based searches that have yielded additional NGF/BDNF relatives. Members of the...

The same receptor tyrosine kinase (RTK) can mediate strikingly different biological responses in a fibroblast as opposed to a neuron. We have compared the rapidly induced tyrosine phosphorylations mediated by various RTKs in both NIH3T3 fibroblasts and in the PC12 neuronal precursor cell line and found that each RTK induces a distinct pattern of protein tyrosine phosphorylations in the two cell types. These findings are consistent with a model in which various cell types present a given RTK with different menus of signal transduction components, allowing the same RTK to elicit fundamentally distinct biological responses. Although there are obvious overlaps in the tyrosine phosphorylations induced by different RTKs in the same cell, there are also clear differences. The attempt to dissect these differences revealed that the kinase inhibitors K-252a and staurosporine inhibit RTK autophosphorylation and thus the biological consequences of receptor/ligand interaction. These inhibitors displayed substantially greater specificity for a subset of RTKs (including the neurotrophin receptors) than for other RTKs and acted as remarkably selective blockers of neurotrophin action in both neuronal and nonneuronal cells. A potential therapeutic application for these inhibitors is discussed.

The neurotrophins (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], neurotrophin-3 [NT-3], and neurotrophin-4 [NT-4]) have been defined by their ability to support neuronal survival. These factors utilize the Trk family of receptor tyrosine kinases, perhaps in conjunction with a second component known as the low affinity NGF receptor (LNGFR). Here we demonstrate that TrkB mediates survival and proliferation in response to both BDNF and NT-3 when expressed in a particular strain of NIH 3T3 fibroblasts, with BDNF the more potent ligand. Furthermore, the BDNF dose dependency displayed by these TrkB-expressing fibroblasts is similar to that required to support the survival of primary neurons. The LNGFR is not expressed in our fibroblast system, implying that this receptor is not essential for responses to physiological concentrations of the neurotrophins. We discuss our findings in the context of neurotrophin signaling pathways and mechanisms of neuronal cell death.

We previously identified a protein related to the expression of the ilvGMEDA cluster of Escherichia coli K-12. It was observed that this ilv-related protein was produced at higher levels in UV irradiated cells infected with lambda dilvGMEDA phage with specific ilvG mutations (ValR), compared to phage carrying the wild-type(ValS) ilvG allele. The gene encoding this protein was further localized to a region between rrnC and ilvGMEDA by analyzing restriction fragment subsets in maxicells. We have now determined the nucleotide (nt) sequence of the 3.5-kb segment between rrnC and ilvGMEDA, and two open reading frames (ORFs) are present in the region expected to contain the ilv-related gene. These ORFs predicts Mrs of 18,751 (ORFI) and 20,085 (ORFII) Da, and both ORFs have a strong probability to encode proteins based on codon frequency analysis. Maxicell analysis revealed that a 1319-bp HindIII-SmaI fragment containing ORFI encodes the ilv-related peptide. We deleted a ClaI fragment that removed a portion of ORFI encoding the C-terminal region of the peptide, and maxicell analysis revealed a decrease in the size of the protein produced in accord with the prediction. RNA slot blots and Northern blots were used to characterize transcripts encoding ORFI. A transcript initiated 112 nt from the ilvGp2 promoter, but proceeding in the opposite direction, may encode the ORFI peptide.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, α-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with α-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to α-galactosidase A, and its expression is repressed by tryptophan and induced by 3-β-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using α-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside.

We have isolated and characterized a human genomic clone for a lysosomal enzyme gene. The start point of transcription was identified using primer extension of poly(A)+ mRNA. This genomic clone is specific for human alpha-galactosidase A, and it includes sequences for the promoter, complete signal peptide, first exon, and part of the first intron. Direct and inverted repeat elements of 10, 11, 16, 19, and 22 nucleotides (nt) flank the promoter site. A (GA)n repeat element of approx. 60 nt with strong homology to similar elements identified in several species is located upstream from the promoter. A GGGCGG site specific for DNA-binding protein Sp1 is located near a CAAT box, and the CCGCCC inverted repeat of the Sp1 binding sequence is located by the TATA box. The sequence immediately flanking the ATG start codon of the human alpha-galactosidase A is highly homologous to sequences flanking the ATG start codons of the other human lysosomal hydrolases for which sequence information is available (beta-glucocerebrosidase, cathepsin B, cathepsin D, and beta-hexosaminidase alpha chain), but not for any of the other 133 human signal peptides examined. Our analysis also reveals that conversion of the propeptide to the mature enzyme involves cleavage of a C-terminal rather than an N-terminal fragment. This information about the normal alpha-galactosidase A gene will be useful for comparison to data obtained from patients with Fabry disease, who are characterized by a deficiency of this enzyme. This is the first genomic clone described to date for any lysosomal enzyme, and it establishes a reference for future analyses of the molecular events that mediate the expression of this important class of enzymes.

The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.