Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells

Lu, Hsieng S.; Clogston, Christi L.; Wypych, Jette; Parker, Vann P.; Lee, Terry D.; Swiderek, K. M.; Baltera, Robert F.; Patel, Avantika C.; Chang, David C.; Brankow, David; Liu, Xiaodong; Ogden, Steven G.; Karkare, Subhash B.; Hu, Sylvia; Zsebo, Krisztina M.; Langley, Keith

doi:10.1016/0003-9861(92)90106-7

Cited by 36 publications

(20 citation statements)

References 24 publications

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“…The soluble cleavage product of KLMl migrated slightly more slowly, consistent with a predominant cleavage site after alanine-165. This result with mouse KL is consistent with a recent report identifying alanine-165 as the predominant cleavage site of the KL-Mi sequence from humans (Lu et al, 1992). In repeated experiments where soluble KL derived from KL-M2 was run in adjacent lanes with the soluble KL derived from KL-M1, the apparent size difference was estimated at 2400 Da (Figure 6), indicating that the KL-M2 cleavage site is at or near serine-142.…”

Section: Proteolytic Cleavage Does Not Occur In Kl Peptidessupporting

confidence: 79%

Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane.

Cheng

Flanagan

1994

MBoC

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The kit ligand (KL) is one of several growth factors that are active as transmembrane molecules and can also be proteolytically cleaved to yield soluble forms. We have investigated the signals and structural determinants that control the cleavage of KL. Presentation at the membrane appears to be critical, because no cleavage occurs in variants that lack a transmembrane domain. Signals in the cytoplasmic domain do not seem to be required, because cleavage was not blocked by removal of the C-terminal valine residue, deletion of the whole cytoplasmic tail, or the replacement of the cytoplasmic tail that occurs in the Sl17H mutation. KL thus appears to differ from transforming growth factor-alpha, which apparently requires a C-terminal valine as a signal for cleavage. Although proteolysis must be tightly restricted to the correct cell surface proteins and sites within each protein, cleavage of KL does not seem to be determined entirely by a requirement for a specific substrate sequence. However, the effects of deletion or insertion variants of KL suggest that cleavage may be limited to sites within a specific range of distances from the cell membrane.

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Section: Proteolytic Cleavage Does Not Occur In Kl Peptidessupporting

confidence: 79%

Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane.

Cheng

Flanagan

1994

MBoC

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“…Other cytokines and chemokines such as IL-2, RANTES, GM-CSF, and IL-6 have been found by subcellular fractionation and confocal or electron microscopic analysis to reside in the eosinophil secretory granules. 28,29,41,42 This observation suggests that these pre-stored molecules and, hence, SCF can be released together with MBP and other granule-associated mediators in a prompt fashion on eosinophil activation. Although incubation with PMA and calcium ionophore-potent stimuli for eosinophil degranulation 43 and SCF cleavage 40 -induced eosinophil peroxidase (EoP) release (data not shown), measurable SCF secretion from the eosinophils was not detected.…”

Section: Discussionmentioning

confidence: 93%

“…When COS-1 cells were transfected with the shorter SCF isoform, they displayed specific protein products of 28 and 32 kd. 2 In addition, Lu et al 41 showed that COS-1 cells transfected with the larger SCF cDNA presented 3 glycosylated bands of 28, 35, and 40 kd. After complete deglycosylation, the molecular weight was 18 to 19 kd.…”

Section: Discussionmentioning

confidence: 98%

Human peripheral blood eosinophils express stem cell factor

Hartman¹,

Piliponsky²,

Temkin³

et al. 2001

Blood

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Stem cell factor (SCF) or c-Kit ligand is a cytokine associated with the differentiation, survival, and activation of mast cells. Eosinophils have pleiotropic functions in several diseases and, together with mast cells, are key cells in allergy. Mast cell-eosinophil interactions can take place during the late and chronic phases of allergy. It was, therefore, investigated whether eosinophils can produce SCF and consequently influence mast cells. Human peripheral blood eosinophils variably expressed mRNA for the soluble and uncleaved forms of SCF (reverse transcription-polymerase chain reaction) and produced the 18.5-kd protein backbone of SCF (Western blot analysis). After overnight incubation in medium, eosinophils also produced SCF of higher molecular weight (42-45 kd) that might represent its glycosylated forms. Eosinophils expressed cytoplasmic SCF that colocalized with major basic protein (confocal laser microscopy). Freshly isolated eosinophils contained 8.9 +/- 1.7 pg SCF/10(6) (mean +/- SEM; enzyme-linked immunosorbent assay). Although overnight incubation of the eosinophils in either culture medium or in phorbol 12-myristate 13-acetate-calcium ionophore did not cause the secretion of SCF, the addition of chymase induced SCF release. In summary, it was demonstrated that human peripheral blood eosinophils are a source of SCF. These results may contribute to a better understanding of the interactions between eosinophils and mast cells in allergic inflammation.

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“…We have shown previously that the faster-migrating band on SDSIPAGE of glycosylated CHO cell-derived human SCF-( 1 -165) (Fig. 1 B) has N-linked carbohydrate attached at Asnl20, and the more slowly migrating band has N-linked carbohydrate attached at Am120 and Asn65 [14]. Thus, it appears that glycosylation at these sites does not interfere with recognition by mAb 7H6.…”

Section: Discussionmentioning

confidence: 95%

Epitope Mapping and Immunoneutralization of Recombinant Human Stem‐Cell Factor

Mendiaz¹,

Chang²,

Boone³

et al. 1996

European Journal of Biochemistry

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The epitope regions of three anti-[stem-cell factor (SCF)] Ig have been mapped by characterization of immunoreactivities against truncated forms of SCF in imrnunoblots and against synthetic peptides in solution-phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61 -95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31. The ability of the anti-SCF lg to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied. mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured. mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61 -65 and 91 -95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implicatioiis for the structure of S CF.

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Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells

Cited by 36 publications

References 24 publications

Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane.

Transmembrane kit ligand cleavage does not require a signal in the cytoplasmic domain and occurs at a site dependent on spacing from the membrane.

Human peripheral blood eosinophils express stem cell factor

Epitope Mapping and Immunoneutralization of Recombinant Human Stem‐Cell Factor

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