Structural requirements for palmitoylation of surfactant protein C precursor

Pulmonary surfactant protein C (SP-C) propeptide (proSP-C) is a type II transmembrane protein that is palmitoylated on two cysteines adjacent to its transmembrane domain. To study the structural requirements for palmitoylation of proSP-C, His-tagged human proSP-C and mutant forms were expressed in Chinese hamster ovary cells and analysed by metabolic labelling with [3H]palmitate. Mutations were made in the amino acid sequence representing mature SP-C, as deletion of the N- and C-terminal propeptide parts showed that this sequence by itself could already be palmitoylated. Substitution of the transmembrane domain by an artificial transmembrane domain had no effect on palmitoylation. However, an inverse correlation was found between palmitoylation of proSP-C and the number of amino acids present between the cysteines and the transmembrane domain. Moreover, substitution by alanines of amino acids localized on the N-terminal side of the cysteines had drastic effects on palmitoylation, probably as a result of the removal of hydrophobic amino acids. These data, together with the observation that substitution by alanines of the amino acids localized between the cysteines and the transmembrane domain had no effect on palmitoylation, suggest that the palmitoylation of proSP-C depends not on specific sequence motifs, but more on the probability that the cysteine is in the vicinity of the membrane surface. This is probably determined not only by the number of amino acids between the cysteines and the transmembrane domain, but also by the hydrophobic interaction of the N-terminus with the membrane. This may also be the case for the palmitoylation of other transmembrane proteins.

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Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649597 ◽

1993 ◽

Vol 70 (03) ◽

pp. 418-422 ◽

Cited By ~ 23

Author(s):

Masaharu Aritomi ◽

Naoko Watanabe ◽

Rika Ohishi ◽

Komakazu Gomi ◽

Takao Kiyota ◽

...

Keyword(s):

Protein C ◽

Antithrombin Iii ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Time Lag ◽

Chinese Hamster ◽

Ovary Cells ◽

Soluble Thrombomodulin ◽

Simulation Based ◽

Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

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The transmembrane domain of surfactant protein C precursor determines the morphology of the induced membrane compartment in CHO cells

European Journal of Cell Biology ◽

10.1078/0171-9335-00320 ◽

2003 ◽

Vol 82 (6) ◽

pp. 285-294 ◽

Cited By ~ 2

Author(s):

Anja ten Brinke ◽

George Posthuma ◽

Joseph J. Batenburg ◽

Henk P. Haagsman ◽

Anja N.J.A. Ridder ◽

...

Keyword(s):

Cho Cells ◽

Protein C ◽

Transmembrane Domain ◽

Surfactant Protein ◽

Induced Membrane ◽

Surfactant Protein C ◽

Membrane Compartment

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Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues

Journal of Cell Science ◽

10.1242/jcs.114.2.293 ◽

2001 ◽

Vol 114 (2) ◽

pp. 293-302

Author(s):

A.F. Kabore ◽

W.J. Wang ◽

S.J. Russo ◽

M.F. Beers

Keyword(s):

Protein C ◽

Transmembrane Protein ◽

A549 Cells ◽

Surfactant Protein ◽

Mutant Protein ◽

Cysteine Residues ◽

Western Blots ◽

Surfactant Protein C ◽

C Terminus ◽

Aggresome Formation

Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1–194)), C-terminally deleted proSP-C (EGFP/SP-C(1–185); EGFP/SP-C(1–191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1–194) and EGFP/SP-C(1–191)were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1–185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (γ)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.

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Structural requirements for palmitoylation of surfactant protein C precursor

Biochemical Journal ◽

10.1042/0264-6021:3610663 ◽

2002 ◽

Vol 361 (3) ◽

pp. 663 ◽

Cited By ~ 6

Author(s):

Anja ten BRINKE ◽

Arie B. VAANDRAGER ◽

Henk P. HAAGSMAN ◽

Anja N.J.A. RIDDER ◽

Lambert M.G. van GOLDE ◽

...

Keyword(s):

Protein C ◽

Surfactant Protein ◽

Structural Requirements ◽

Surfactant Protein C

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Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

Biochemical Journal ◽

10.1042/bj20030301 ◽

2003 ◽

Vol 374 (1) ◽

pp. 27-36 ◽

Cited By ~ 14

Author(s):

Wei CAO ◽

Larry H. MATHERLY

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Substrate Binding ◽

Chinese Hamster ◽

Water Soluble ◽

Cysteine Residues ◽

Reduced Folate Carrier ◽

Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

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