Glucose-6-phosphatase (G6Pase) is the enzyme deficient in glycogen storage disease type 1a, an autosomal recessive disorder. We have previously identified six mutations in the G6Pase gene of glycogen storage disease type 1a patients and demonstrated that these mutations abolished or greatly reduced enzymatic activity of G6Pase, a hydrophobic protein of 357 amino acids. Of these, four mutations (R83C, R295C, G222R, and Q347X) are missense and one (Q347X) generates a truncated G6Pase of 346 residues. To further understand the roles and structural requirements of amino acids 83, 222, 295, and those at the carboxyl terminus in G6Pase catalysis, we characterized mutant G6Pases generated by near-saturation mutagenesis of the aforementioned amino acids. Substitution of Arg-83 with amino acids of diverse structures including Lys, a conservative change, yielded mutant G6Pase with no enzymatic activity. On the other hand, substitution of Arg-295 with Lys (R295K) retained high activity, and R295N, R295S, and R295Q exhibited moderate activity. All other substitutions of Arg-295 had no G6Pase activity, suggesting that the role of Arg-295 is to stabilize the protein either by salt bridge or hydrogen-bond formation. Substitution of Gly-222, however, remained functional unless a basic (Arg or Lys), acidic (Asp), or large polar (Gln) residue was introduced, consistent with the hydrophobic requirement of codon 222, which is predicted to be in the fourth membrane-spanning domain. It is possible that Arg-83 is involved in stabilizing the enzyme (His)-phosphate intermediate formed during G6Pase catalysis. There exist 9 conserved His residues in human G6Pase. His-9, His-119, His-252, and His-353 reside on the same side of the endoplasmic reticulum membrane as Arg-83. Whereas H119A mutant G6Pase had no enzymatic activity, H9A, H252A, and H353A mutant G6Pases retained significant activity. Substitution of His-119 with amino acids of diverse structures also yielded mutant G6Pase with no activity, suggesting that His-119 is the phosphate acceptor in G6Pase catalysis. We also present data demonstrating that the carboxyl-terminal 8 residues in human G6Pase are not essential for G6Pase catalysis.

Glycogen storage disease (GSD) type la is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type la patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type la patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K3SSSP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis. (J. Clin. Invest. 1994. 93:1994-1999 Key words: inborn error of metabolism -genetic mutation * endoplasmic reticulum and/or retention signal

We have synthesized triple helix forming oligonucleotides (TFOs) that target a psoralen (pso) interstrand crosslink to a specific chromosomal site in mammalian cells. Mutagenesis of the targeted crosslinks results in base substitutions and deletions. Identification of the gene products involved in mutation formation is important for developing practical applications of pso-TFOs, and may be informative about the metabolism of other interstrand crosslinks. We have studied mutagenesis of a pso-TFO genomic crosslink in repair proficient and deficient cells. Deficiencies in non homologous end joining and mismatch repair do not influence mutation patterns. In contrast, the frequency of base substitutions is dependent on the activity of ERCC1/XPF and polymerase ζ, but independent of other nucleotide excision repair (NER) or transcription coupled repair (TCR) genes. In NER/TCR deficient cells the frequency of deletions rises, indicating that in wild-type cells NER/TCR functions divert pso-TFO crosslinks from processes that result in deletions. We conclude that targeted pso-TFO crosslinks can enter genetically distinct mutational routes that resolve to base substitutions or deletions.

The gene encoding the human pregnancy-specific glycoprotein (PSG) belongs to a gene subfamily, comprised of the carcinoembryonic antigen (CEA) and PSG subgroups, within the immunoglobulin superfamily. To study the functional roles of PSG during development in an animal model, we isolated and characterized a near full-length cDNA (rnCGM6) encoding a PSG-related protein from a rat placental cDNA library. rnCGM6 is 2,068 bp in length and contains an open reading frame that encodes a 475-amino-acid polypeptide with a predicted molecular mass of 53 kD. The 5' noncoding sequence is 173 nucleotides, and primer-extension experiments demonstrate that the transcriptional initiation site is located 22-24 nucleotides further upstream. The 3' noncoding sequence contains 470 nucleotides which is followed by a poly(A) tail. In contrast to human PSGs, which contain one immunoglobulin variable-like and two to three immunoglobulin constant-like protein domains, rnCGM6 contains three immunoglobulin variable-like domains and one immunoglobulin constant-like domain. rnCGM6 contains six potential N-linked glycosylation sites and, in its carboxyl-terminal domain, a tyrosine protein kinase phosphorylation site. The tyrosine phosphorylation site is conserved among all rat and human PSG members. rnCGM6 hybridized with a major 2.5-kb and two minor 3.0- and 3.5-kb mRNAs, all primarily expressed in the rat placenta. Ribonuclease protection analysis, using probes specific to the 5', middle, and 3' regions of rnCGM6, and the 5' region of a previously identified cDNA, rnCGM1, mainly yielded fully-protected fragments indicating relatively low sequence similarity among rat PSG-related proteins. Northern hybridization and ribonuclease protection assays also suggest that rnCGM6 may be the major PSG member in rat.