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Jeffrey J Gorman

Publications and source records attributed to Jeffrey J Gorman.

12 recordsLinked to original sources
PubMed2007

The location and characterisation of the O-linked glycans of the human insulin receptor.

O-linked glycosylation is a post-translational and post-folding event involving exposed S/T residues at beta-turns or in regions with extended conformation. O-linked sites are difficult to predict from sequence analyses compared to N-linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O-glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin-type O-glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763- correctly, T756- incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor beta-chain. This region which also includes N-linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765-770, the known linear epitope for the monoclonal antibody 18-44.

Acetylgalactosamine↗
PubMed2006

Proteomic analysis of mouse growth plate cartilage.

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.

Animals↗
PubMed2006

Post-translational modification accounts for the presence of varied forms of nerve growth factor in Australian elapid snake venoms.

The Australian elapid snakes are amongst the most venomous snakes in the world, but much less is known about the overall venom composition in comparison to Asian and American snakes. We have used a combined approach of cDNA cloning and 2-DE with MS to identify nerve growth factor (NGF) in venoms of the Australian elapid snakes and demonstrate its neurite outgrowth activity. While a single 730 nucleotide ORF, coding for a 243 amino acid precursor protein was detected in all snakes, use of 2-DE identified NGF proteins with considerable variation in molecular size within and between the different snakes. The variation in size can be explained at least in part by N-linked glycosylation. It is possible that these modifications alter the stability, activity and other characteristics of the snake NGFs. Further characterisation is necessary to delineate the function of the individual NGF isoforms.

Amino Acid Sequence↗
PubMed2005

Molecular diversity in venom from the Australian Brown snake, Pseudonaja textilis.

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A2s, and pre- and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.

Animals↗
PubMed2005

LH3, a "homologue" of the mastadenoviral E1B 55-kDa protein is a structural protein of atadenoviruses.

Ovine adenovirus serotype 7 (OAdV), the prototype atadenovirus, has gene homologues for most mastadenovirus structural proteins but lacks proteins V and IX. Instead, OAdV has structural proteins of 32 and 42 kDa although the gene encoding the latter had not previously been identified. The presently reported studies of OAdV virions have now identified a minor structural polypeptide of approximately 40 kDa as the product of the L1 52/55-kDa gene and, more surprisingly, shown that the 42-kDa protein is encoded by LH3. This gene product was previously thought to be a homologue of mastadenovirus E1B 55 kDa, which is a multi-functional, non-structural protein that cooperates with E1A in cell transformation. The lack of transforming activity previously demonstrated for OAdV combined with a structural role for the LH3 product indicates that the protein has a different function in atadenoviruses. We discuss the abundance and likely core location of LH3 in the virion and the possible derivation of the E1B 55-kDa gene from the LH3 gene.

Adenovirus E1B Proteins↗
PubMed2004

Determination of the disulfide bond arrangement of dengue virus NS1 protein.

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.

Amino Acid Sequence↗
PubMed2003

A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition.

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Alleles↗
PubMed2003

Oxygen-dependent regulation of hypoxia-inducible factors by prolyl and asparaginyl hydroxylation.

To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, maintaining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and physiological responses to adapt to changes in oxygen availability. In the past few years it has become evident that the transcriptional protein complex hypoxia-inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen-dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.

Asparagine↗
PubMed2002

FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O(2) to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway.

Amino Acid Sequence↗
PubMed2002

Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch.

The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.

Amino Acid Sequence↗
PubMedDate not supplied

Protein disulfide bond determination by mass spectrometry.

The determination of disulfide bonds is an important aspect of gaining a comprehensive understanding of the chemical structure of a protein. The basic strategy for obtaining this information involves the identification of disulfide-linked peptides in digests of proteins and the characterization of their half-cystinyl peptide constituents. Tools for disulfide bond analysis have improved dramatically in the past two decades, especially in terms of speed and sensitivity. This improvement is largely due to the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), and complementary analyzers with high resolution and accuracy. The process of pairing half-cystinyl peptides is now generally achieved by comparing masses of non-reduced and reduced aliquots of a digest of a protein that was proteolyzed with intact disulfide bonds. Pepsin has favorable properties for generating disulfide-linked peptides, including its acidic pH optimum, at which disulfide bond rearrangement is precluded and protein conformations are likely to be unfolded and accessible to cleavage, and broad substrate specificity. These properties potentiate cleavage between all half-cystine residues of the substrate protein. However, pepsin produces complex digests that contain overlapping peptides due to ragged cleavage. This complexity can produce very complex spectra and/or hamper the ionization of some constituent peptides. It may also be more difficult to compute which half-cystinyl sequences of the protein of interest are disulfide-linked in non-reduced peptic digests. This ambiguity is offset to some extent by sequence tags that may arise from ragged cleavages and aid sequence assignments. Problems associated with pepsin cleavage can be minimized by digestion in solvents that contain 50% H(2) (18)O. Resultant disulfide-linked peptides have distinct isotope profiles (combinations of isotope ratios and average mass increases) compared to the same peptides with only (16)O in their terminal carboxylates. Thus, it is possible to identify disulfide-linked peptides in digests and chromatographic fractions, using these mass-specific markers, and to rationalize mass changes upon reduction in terms of half-cystinyl sequences of the protein of interest. Some peptides may require additional cleavages due to their multiple disulfide bond contents and/or tandem mass spectrometry (MS/MS) to determine linkages. Interpretation of the MS/MS spectra of peptides with multiple disulfides in supplementary digests is also facilitated by the presence of (18)O in their terminal carboxylates.

Amino Acid Sequence↗