Physician/Scientist

Genetics

The Molecular Genetics of Acid Sphingomyelinase (ASM)

The Human ASM cDNA

In 1989, the first cDNAs encoding human ASM were isolated. Two distinct ASM cDNAs were identified (designated "types 1 and 2") whose predicted amino acid sequences were colinear with peptide amino acid sequences determined from purified human urinary ASM. The type 1 cDNA contained a unique 172-bp sequence encoding 57 amino acids that was replaced in the type 2 cDNA by a 40-bp sequence encoding 13 different amino acids. From extensive library screenings, a third type of ASM cDNA also was isolated and sequenced (designated "type 3"). Although this cDNA was colinear with the ASM amino acid sequences, it did not contain the type 1- or 2-specific sequences. In addition, the reading frame of the type 3 cDNA was altered, and a premature stop codon was identified at codon 248. Northern hybridization, RNase protection, and PCR amplification analyses documented the occurrence of full-length type 1, 2, and 3 mRNAs and suggested that the three transcripts represented about 90, 10, and 1 percent of the total cellular ASM mRNA, respectively, in fibroblasts and placenta. To investigate their functional integrity, the full-length types 1, 2, and 3 cDNAs were individually subcloned into the mammalian expression vector, p91023(B), and transiently expressed in COS-1 cells. Only the full-length type 1 cDNA expressed catalytically active enzyme. The fact that the types 2 and 3 cDNA did not express catalytically active enzymes suggested that these mRNAs were nonfunctional and resulted from aberrant splicing of the ASM hnRNA - a hypothesis that was confirmed by analysis of the ASM genomic sequence (see "The Human ASM Gene").

The 2347-bp full-length type 1 cDNA had an open reading frame (ORF) of 1890 bp encoding 629 amino acids. The size of the full-length type 1 cDNA was consistent with the occurrence of the ~2.5-kb transcript observed in the northern hybridization experiments, the ~150-bp difference presumably a result of the upstream 5' untranslated sequences and the length of the polyA tract. Notably, two in-frame initiation codons were identified within the 5' region. Since the purified enzyme's N-terminus was blocked, it was not known which of these potential initiation codons was utilized in vivo. However, site-directed mutagenesis and transient expression studies demonstrated that both initiation sites could function in vitro. Using the von Heijne weight-matrix method, the optimal signal peptide cleavage of the ASM precursor polypeptide was predicted to occur after amino acid residue 46. The 14 amino acids preceding the signal peptide cleavage site had a particularly hydrophobic core consisting of five leucine/alanine repeats encoded by a CTGGCG hexanucleotide sequence. Six N-N-glycosylation sites were predicted in the mature ASM polypeptide, and it is now known that five of these sites are utilized (Ferlinz et al. 1995).

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The Human ASM Gene

Genomic Structure and the Nature of Alternative Splicing
The complete ASM genomic region, including 1116 and 468 of 5' and 3' untranslated nucleotides, respectively, has been isolated and sequenced. This housekeeping gene is about 5 kb long and is composed of six exons ranging in size from 77 to 773 bp and five introns ranging in size from 153 to 1059 bp. Exon 2 is unusually large, encoding 258 amino acids, or about 44 percent of the mature ASM polypeptide. Immediately downstream from the type 2-specific sequence, within intron 2, is a single Alu 1 repeat element inserted in the reverse orientation. The Alu 1 element was placed into the "a branch" according to the classification of Jurka and Smith, indicating the ancestral nature of the ASM gene. The regulatory region upstream of the ASM coding sequence was GC-rich and contained putative promoter elements, including SP1, TATA, CAAT, NF-1, and AP-1 binding sites.

Analysis of the genomic sequence indicated that alternative splicing of the ASM hnRNA was the molecular basis for the types 1, 2, and 3 transcripts. The type 1-specific 172 bp sequence was encoded by exon 3, whereas the type 2-specific 40 bp sequence was located at the 5' end of intron 2, followed by a cryptic donor splice site (aag gtgaat). Furthermore, there was a poor donor splice site (AAA gtgagg) at the junction of exon 3 and intron 3. Thus, the occurrence of the type 2 and 3 ASM transcripts resulted from the fact that in about 10 percent of the hnRNAs, the normal donor site was not functional, and splicing proceeded either to the cryptic donor splice site or to the next donor site. A G-to-A transition of the nucleotide immediately adjacent to the invariant gt consensus dinucleotide in the normal donor splice site may cause these alternative splicing events, since this alteration has been implicated as the cause of abnormal splicing in the pro a1 (I) collagen gene resulting in Ehlers-Danlos syndrome type VII.

The ASM genomic region encoded three other long ORF that predicted polypeptides of 101, 104, and 158 amino acid residues, respectively. The transcriptional orientations of ORF 1 and ORF 2 were opposite those of ASM, and the predicted proteins shared no homology with ASM or any other proteins in the Swiss-Prot protein database. In contrast, ORF 3 was in the same transcriptional orientation and coding phase as the ASM gene. This ORF began within intron 2, overlapped ASM exon 3, and extended into intron 3. No known functions for these ORFs have been described.

Regional Mapping of the Human ASM Gene
Using molecular techniques, the locus for the human ASM gene was assigned to the chromosomal region 11p15.1 to p15.4. Selective PCR amplification of a human exonic sequence and in situ hybridization of the radiolabeled cDNA provided independent data that assigned the gene locus (designated SMPD-1) to this narrow region on chromosome 11. Although a number of other sphingomyelinases and related phospholipases have been identified, these molecular studies identified only a single locus for human ASM, indicating the absence of homologous coding sequences and pseudogenes elsewhere in the genome. In addition, genomic Southern blotting experiments were consistent with a single ASM gene. Two other lysosomal genes have been assigned to 11p,- -cathepsin D to 11p15 and acid phosphatase 2 to 11p11.

The results of these studies corrected a previous provisional assignment of the human ASM gene to chromosome 17. The reason for this discrepancy is not known, although it is possible that the artificial substrate (i.e., 2-hexadecanoyl- amino-4-nitrophenol phosphorylcholine) used in the earlier study may not have been specific for ASM, particularly under the thermo-inactivation conditions used.

Polymorphisms Within the ASM Gene
Two common polymorphisms have been identified within the ASM gene leading to amino acid substitutions at codons 322 and 506. The common allele for each codon was Thr 322 (ACA) and Gly 506 (GGG) (allele frequencies of 0.6 and 0.8, respectively). The less common alleles were Ile 322 (ATA) and Arg 506 (AGG). Notably, the Gly 506 polymorphism creates a new MspI restriction site, facilitating its identification by restriction enzyme analysis. In addition to these polymorphisms, the number of alanine/leucine repeats within the ASM signal peptide region also has been shown to be polymorphic (Wan et al.). These common polymorphisms should be useful markers for the orientation of anonymous polymorphic sequences and/or sequence tagged sites in the chromosomal region 11p15.

The Molecular Genetics of Murine ASM
The full-length human ASM cDNA has been used to isolate the full-length cDNA encoding murine ASM. The full-length murine ASM cDNA was 2320 bp and contained an 1884-bp ORF encoding 627 amino acids. Transient expression in COS-1 cells demonstrated the functional integrity of the murine cDNA. Overall, the nucleotide and amino acid identities between the human and murine ASM sequences were about 81 and 83 percent, respectively. Notably, five of the six predicted N-N-glycosylation sites in human ASM were conserved in the mouse sequence. This observation was consistent with previous biochemical and molecular data, which indicated that only five of the N-glycosylation sites in the human polypeptide were used (Ferlinz et al. 1995).

The full-length murine ASM cDNA has been used to isolate the complete genomic region encoding murine ASM (Newrzella and Stoffel, 1992). Similar to the human gene, the mouse ASM coding region was divided among six exons. The position and relative size of the ASM introns and exons were highly conserved between mice and humans, with the notable exception that intron 2 in the human gene was 1059 bp, whereas in the murine gene it was 510 bp. This observation was consistent with what transpired when an Alu 1 repetitive element was inserted into intron 2 of the human gene. As in the human gene, there was a poor donor splice site adjacent to exon 3 in the mouse gene. However, the cryptic donor splice site in intron 2 of the human gene was not conserved in the mouse sequence. Each of the other splice donor and acceptor sites in the murine ASM gene adhered to the consensus sequences.

In order to determine the chromosomal location of the murine ASM gene, a panel of nine mouse/hamster somatic-cell hybrids was analyzed using a PCR amplification detection assay (Horinouchi et al. 1993). Analysis of the somatic-cell hybrid panel revealed that the presence of the murine ASM gene was 100 percent concordant with the presence of mouse chromosome 7. This finding was consistent with the assignment of the human ASM gene to 11p15.1-p15.4, since the short arm of human chromosome 11 and mouse chromosome 7 are syntenic.M

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