scholarly journals A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

2021 ◽  
Author(s):  
Saulius Vainauskas ◽  
Hélène Guntz ◽  
Elizabeth McLeod ◽  
Colleen McClung ◽  
Cristian Ruse ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Phosphatase ◽  
Protein Sequence ◽  
High Selectivity ◽  
Granulocyte Colony Stimulating Factor ◽  
Peptide Substrate ◽  
Significant Progress ◽  
One Step ◽  
Stimulating Factor ◽  
Sequence Bias

AbstractAnalysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

scholarly journals Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation

1993 ◽  
Vol 13 (12) ◽  
pp. 7774-7781
Author(s):  
F Dong ◽  
C van Buitenen ◽  
K Pouwels ◽  
L H Hoefsloot ◽  
B Löwenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Splice Variant ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor Cells ◽  
Carboxy Terminal ◽  
Stimulating Factor

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

Inhibition of granulocyte colony-stimulating factor–mediated myeloid maturation by low level expression of the differentiation-defective class IV granulocyte colony-stimulating factor receptor isoform

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3335-3340 ◽  
Author(s):  
Scott M. White ◽  
Mark H. Alarcon ◽  
David J. Tweardy
Keyword(s):  
Amino Acids ◽  
Signaling Pathways ◽  
Bone Marrow Cells ◽  
Class I ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Mrna Isoforms ◽  
Murine Cell Line ◽  
Stimulating Factor ◽  
Class Iv

Abstract In acute myeloid leukemia (AML), granulocyte colony-stimulating factor receptor (G-CSFR) proliferative and maturational signaling pathways are uncoupled. Seven human G-CSFR mRNA isoforms exist, named class I through class VII. The 183-amino acid cytosolic domain of the class I isoform provides all signaling activities. The class IV isoform is “differentiation defective” because the carboxy-terminal 87 amino acids are replaced with 34 amino acids of novel sequence. In more than 50% of AML samples, the class IV/class I G-CSFR mRNA ratio is aberrantly elevated compared to normal CD34+ bone marrow cells. We hypothesized that the increased relative expression of class IV G-CSFR in AML uncouples proliferative and maturational G-CSFR signaling pathways. To test this, we transfected the G-CSF–responsive murine cell line 32Dcl3 with class IV G-CSFR cDNA. After 10 days of G-CSF stimulation, clones expressing class IV G-CSFR had greater percentages of myeloblasts and promyelocytes than controls (53% ± 13% versus 3% ± 2%). Differential counts over time demonstrated delayed G-CSF–driven maturation in 5 class IV-expressing clones, with 2 clones demonstrating a subpopulation that completely failed to differentiate. Heterologous class IV expression did not affect G-CSF–dependent proliferation. Class IV/murine G-CSFR mRNA ratios after 24 hours of G-CSF stimulation for 3 of the 5 clones (range, 0.090 to 0.245; mean, 0.152 ± 0.055) are within the range of class IV/class I mRNA ratios seen in patients with AML. This indicates that aberrantly increased relative class IV G-CSFR expression seen in AML can uncouple G-CSFR proliferative and maturational signaling pathways.

Inhibition of granulocyte colony-stimulating factor–mediated myeloid maturation by low level expression of the differentiation-defective class IV granulocyte colony-stimulating factor receptor isoform

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3335-3340 ◽  
Author(s):  
Scott M. White ◽  
Mark H. Alarcon ◽  
David J. Tweardy
Keyword(s):  
Amino Acids ◽  
Signaling Pathways ◽  
Bone Marrow Cells ◽  
Class I ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Mrna Isoforms ◽  
Murine Cell Line ◽  
Stimulating Factor ◽  
Class Iv

In acute myeloid leukemia (AML), granulocyte colony-stimulating factor receptor (G-CSFR) proliferative and maturational signaling pathways are uncoupled. Seven human G-CSFR mRNA isoforms exist, named class I through class VII. The 183-amino acid cytosolic domain of the class I isoform provides all signaling activities. The class IV isoform is “differentiation defective” because the carboxy-terminal 87 amino acids are replaced with 34 amino acids of novel sequence. In more than 50% of AML samples, the class IV/class I G-CSFR mRNA ratio is aberrantly elevated compared to normal CD34+ bone marrow cells. We hypothesized that the increased relative expression of class IV G-CSFR in AML uncouples proliferative and maturational G-CSFR signaling pathways. To test this, we transfected the G-CSF–responsive murine cell line 32Dcl3 with class IV G-CSFR cDNA. After 10 days of G-CSF stimulation, clones expressing class IV G-CSFR had greater percentages of myeloblasts and promyelocytes than controls (53% ± 13% versus 3% ± 2%). Differential counts over time demonstrated delayed G-CSF–driven maturation in 5 class IV-expressing clones, with 2 clones demonstrating a subpopulation that completely failed to differentiate. Heterologous class IV expression did not affect G-CSF–dependent proliferation. Class IV/murine G-CSFR mRNA ratios after 24 hours of G-CSF stimulation for 3 of the 5 clones (range, 0.090 to 0.245; mean, 0.152 ± 0.055) are within the range of class IV/class I mRNA ratios seen in patients with AML. This indicates that aberrantly increased relative class IV G-CSFR expression seen in AML can uncouple G-CSFR proliferative and maturational signaling pathways.

scholarly journals Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.

1993 ◽  
Vol 13 (4) ◽  
pp. 2384-2390 ◽  
Author(s):  
S F Ziegler ◽  
T A Bird ◽  
K K Morella ◽  
B Mosley ◽  
D P Gearing ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatoma Cell ◽  
Signal Transduction Pathway ◽  
Cytoplasmic Domain ◽  
Sequence Motifs ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Human Hepatoma Cell ◽  
Functional Region ◽  
Stimulating Factor

Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.

scholarly journals Expression cloning of a human granulocyte colony-stimulating factor receptor: a structural mosaic of hematopoietin receptor, immunoglobulin, and fibronectin domains.

1990 ◽  
Vol 172 (6) ◽  
pp. 1559-1570 ◽  
Author(s):  
A Larsen ◽  
T Davis ◽  
B M Curtis ◽  
S Gimpel ◽  
J E Sims ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Surface Proteins ◽  
Expression Cloning ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hybridization Probe ◽  
Extracellular Region ◽  
Fibronectin Domains ◽  
Stimulating Factor

We report the isolation from a placental library, of two cDNAs that can encode high affinity receptors for granulocyte colony-stimulating factor (G-CSF) when expressed in COS-7 cells. The cDNAs are predicted to encode integral membrane proteins of 759 and 812 amino acids in length. The predicted extracellular and membrane spanning sequences of the two clones are identical, as are the first 96 amino acids of their respective cytoplasmic regions. Different COOH termini of 34 or 87 residues are predicted for the two cDNAs, due apparently to alternate splicing. The receptor with the longer cytoplasmic domain is the closest human homologue of the murine G-CSF receptor recently described by Fukunaga et al. (Fukunaga, R., E. Ishizaka-Ikeda, Y. Seto, and S. Nagata. 1990. Cell. 61:341). A hybridization probe derived from the placental G-CSF receptor cDNA detects a approximately 3-kb transcript in RNAs isolated from placenta and a number of lymphoid and myeloid cells. The extracellular region of the G-CSF receptors is composed of four distinct types of structural domains, previously recognized in other cell surface proteins. In addition to the two domains of the HP receptor family-defining region (Patthy, L. 1990. Cell. 61:13) it incorporates one NH2-terminal Ig-like domain, and three additional repeats of fibronectin type III-like domains. The presence of both an NH2-terminal Ig-like domain and multiple membrane-proximal FN3-like domains suggests that the G-CSF receptor may be derived from an ancestral NCAM-like molecule and that the G-CSF receptor may function in some adhesion or recognition events at the cell surface in addition to the binding of G-CSF.

scholarly journals Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.

1993 ◽  
Vol 13 (12) ◽  
pp. 7774-7781 ◽  
Author(s):  
F Dong ◽  
C van Buitenen ◽  
K Pouwels ◽  
L H Hoefsloot ◽  
B Löwenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Lines ◽  
Splice Variant ◽  
Transmembrane Domain ◽  
Myeloid Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Myeloid Progenitor Cells ◽  
Carboxy Terminal ◽  
Stimulating Factor

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.

scholarly journals Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3117-3126 ◽  
Author(s):  
BR Avalos ◽  
MG Hunter ◽  
JM Parker ◽  
SK Ceselski ◽  
BJ Druker ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Amino Acid ◽  
Tyrosine Phosphorylation ◽  
Cytoplasmic Domain ◽  
Early Event ◽  
Sequence Motif ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.

scholarly journals Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3698-3704 ◽  
Author(s):  
SE Nicholson ◽  
U Novak ◽  
SF Ziegler ◽  
JE Layton
Keyword(s):  
Amino Acids ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Signaling Molecules ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Kinase Activation ◽  
Stimulating Factor

The protein tyrosine kinases JAK1 and JAK2 are phosphorylated tyrosine after the interaction of granulocyte colony-stimulating factor (G-CSF) with its transmembrane receptor. So too is Stat3, a member of the STAT family of transcriptional activators thought to be activated by the JAK kinases. Truncated G-CSF receptor (G-CSF-R) mutants were used to determine the different regions of the cytoplasmic domain necessary for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. We have shown that G-CSF-induced tyrosine phosphorylation and kinase activation of JAK2 requires the membrane proximal 57 amino acids of the cytoplasmic domain. In contrast, maximal Stat3 tyrosine phosphorylation required amino acids 96 to 183 of the G-CSF-R cytoplasmic domain, Stat3 DNA binding could occur with a receptor truncated 96 amino acids from the transmembrane domain and containing a single tyrosine residue, but was reduced in comparison with the full- length receptor. Together with the tyrosine phosphorylation of Stat3, this finding suggests that additional Stat3 does not appear to be required for proliferation. MAP kinase tyrosine phosphorylation correlated with both the proliferative response and JAK2 activation.

scholarly journals Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2148-2153 ◽  
Author(s):  
RM Barge ◽  
JP de Koning ◽  
K Pouwels ◽  
F Dong ◽  
B Lowenberg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Distal Region ◽  
Proximal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Tyrosine Residues ◽  
Membrane Proximal Region ◽  
Stimulating Factor ◽  
Proliferative Signaling ◽  
Factor G

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.

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