Identification of mammalian GPI-anchored proteins based on amino acid propensities in their GPI attachment signal sequences

ABSTRACT Extracellular proteins made by group A Streptococcus(GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed inEscherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.

Download Full-text

A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

The Journal of Cell Biology ◽

10.1083/jcb.201207163 ◽

2012 ◽

Vol 199 (6) ◽

pp. 907-918 ◽

Cited By ~ 38

Author(s):

Steven F. Trueman ◽

Elisabet C. Mandon ◽

Reid Gilmore

Keyword(s):

Amino Acid ◽

Structural Transition ◽

Protein Translocation ◽

Signal Sequence ◽

Bioinformatic Analysis ◽

Critical Event ◽

Wild Type ◽

Polar Amino Acid ◽

Signal Sequences ◽

Channel Opening

A critical event in protein translocation across the endoplasmic reticulum is the structural transition between the closed and open conformations of Sec61, the eukaryotic translocation channel. Channel opening allows signal sequence insertion into a gap between the N- and C-terminal halves of Sec61. We have identified a gating motif that regulates the transition between the closed and open channel conformations. Polar amino acid substitutions in the gating motif cause a gain-of-function phenotype that permits translocation of precursors with marginally hydrophobic signal sequences. In contrast, hydrophobic substitutions at certain residues in the gating motif cause a protein translocation defect. We conclude that the gating motif establishes the hydrophobicity threshold for functional insertion of a signal sequence into the Sec61 complex, thereby allowing the wild-type translocation channel to discriminate between authentic signal sequences and the less hydrophobic amino acid segments in cytosolic proteins. Bioinformatic analysis indicates that the gating motif is conserved between eubacterial and archaebacterial SecY and eukaryotic Sec61.

Download Full-text

Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence inTrypanosoma brucei

Journal of Cell Science ◽

10.1242/jcs.115.4.805 ◽

2002 ◽

Vol 115 (4) ◽

pp. 805-816 ◽

Cited By ~ 3

Author(s):

Ulrike Böhme ◽

George A. M. Cross

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Surface ◽

Signal Sequence ◽

Surface Expression ◽

Cell Surface Expression ◽

Gpi Anchor ◽

Signal Sequences ◽

Hydrophobic Region ◽

High Conservation

The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence,which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The ω amino acid, to which GPI is transferred, is either Ser,Asp or Asn, the ω+2 amino acid is always Ser, and the ω+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions ω+1, ω+2 and ω+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99%and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring,undetectable in our experiments, might have influenced the evolution of VSG GPI signal sequences.

Download Full-text

The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142372 ◽

1986 ◽

Vol 44 ◽

pp. 150-151

Author(s):

M.K. Lamvik ◽

L.L. Klatt

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Staining Pattern ◽

Banding Patterns ◽

Mass Thickness ◽

New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

Download Full-text

Electron probe x-ray microanalysis and electron microscopy of amino acid absorption and transport by the small intestine: inhibition by chemical poisons and correlation to the elemental composition of the epithelial cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142311 ◽

1986 ◽

Vol 44 ◽

pp. 134-135

Author(s):

A. J. Tousimis

Keyword(s):

Small Intestine ◽

Amino Acid ◽

Epithelial Cells ◽

Elemental Composition ◽

Isotope Labeling ◽

Structural Components ◽

X Ray ◽

Human Small Intestine ◽

Transport Studies

The elemental composition of amino acids is similar to that of the major structural components of the epithelial cells of the small intestine and other tissues. Therefore, their subcellular localization and concentration measurements are not possible by x-ray microanalysis. Radioactive isotope labeling: I131-tyrosine, Se75-methionine and S35-methionine have been successfully employed in numerous absorption and transport studies. The latter two have been utilized both in vitro and vivo, with similar results in the hamster and human small intestine. Non-radioactive Selenomethionine, since its absorption/transport behavior is assumed to be the same as that of Se75- methionine and S75-methionine could serve as a compound tracer for this amino acid.

Download Full-text

Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147776 ◽

1993 ◽

Vol 51 ◽

pp. 388-389

Author(s):

Chi-Ming Wei ◽

Margaret Hukee ◽

Christopher G.A. McGregor ◽

John C. Burnett

Keyword(s):

Amino Acid ◽

Natriuretic Peptide ◽

Vascular Tone ◽

Cardiac Tissue ◽

Ring Structure ◽

Terminal Amino Acid ◽

Amino Acid Peptide ◽

Normal Human ◽

Terminal Amino ◽

The Brain

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.

Download Full-text