Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization.

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Chemical cleavage of proteins on membranes.

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins bound to membranes. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS-skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid-proline (Asp-Pro) peptide bonds; hydroxylamine cleaves at asparagine-glycine (Asn-Gly) peptide bonds, and 2-nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides. In addition, Alternate Protocol an describes CNBr cleavage of PVDF-bound protein previously analyzed by Edman degradation. Finally, a Support Protocol discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.

Decay accelerating activity of complement receptor type 1 (CD35). Two active sites are required for dissociating C5 convertases.

The goal of this study was to identify the site(s) in CR1 that mediate the dissociation of the C3 and C5 convertases. To that end, truncated derivatives of CR1 whose extracellular part is composed of 30 tandem repeating modules, termed complement control protein repeats (CCPs), were generated. Site 1 (CCPs 1-3) alone mediated the decay acceleration of the classical and alternative pathway C3 convertases. Site 2 (CCPs 8-10 or the nearly identical CCPs 15-17) had one-fifth the activity of site 1. In contrast, for the C5 convertase, site 1 had only 0.5% of the decay accelerating activity, while site 2 had no detectable activity. Efficient C5 decay accelerating activity was detected in recombinants that carried both site 1 and site 2. The activity was reduced if the intervening repeats between site 1 and site 2 were deleted. The results indicate that, for the C5 convertases, decay accelerating activity is mediated primarily by site 1. A properly spaced site 2 has an important auxiliary role, which may involve its C3b binding capacity. Moreover, using homologous substitution mutagenesis, residues important in site 1 for dissociating activity were identified. Based on these results, we generated proteins one-fourth the size of CR1 but with enhanced decay accelerating activity for the C3 convertases.

Hemoglobin Raleigh as the cause of a falsely increased hemoglobin A1C in an automated ion-exchange HPLC method.

Irreversible glycation of the hemoglobin A0 (HbA0) beta chain leads to the production of HbA1C, which can be used to monitor long-term blood glucose control in patients with diabetes mellitus. HbA1C is less positively charged than nonglycated HbA0, and this decrease in charge is the basis of ion-exchange and electrophoretic methods that measure HbA1C. We recently identified a sample that appeared to contain 46% HbA1C by an automated ion-exchange HPLC method (Bio-Rad Variant) but only 3.8% by an immunoinhibition latex agglutination method. A combination of traditional and mass spectrometric protein analysis and genomic DNA analysis of the Hb beta chain and genes revealed that the patient was heterozygotic for Hb-Raleigh, a variant containing a valine-->alanine substitution at position 1 of the beta chain. The amino-terminal alanine in this variant Hb is posttranslationally modified by acetylation, leading to a charge difference similar to glycation and making the behavior of HbA1C and Hb Raleigh virtually identical in the ion-exchange HPLC method. This observation suggests that it is important to confirm HbA1C values in excess of 15%, especially if they are not consistent with the clinical picture, by an independent HbA1C method such as immunoassay or boronic acid affinity chromatography. However, for this particular variant Hb, even these latter methods might be misleading, because the acetylated N-terminal amino acid of the Hb-Raleigh beta chain cannot be glycated.

Troponin I phosphorylation in the normal and failing adult human heart.

BACKGROUND: In the failing human heart myofibrillar calcium sensitivity of tension development is greater and maximal myofibrillar ATPase activity is less than in the normal heart. Phosphorylation of the cardiac troponin I (cTnI)-specific NH2-terminus decreases myofilament sensitivity to calcium, while phosphorylation of other cTnI sites decreases maximal myofibrillar ATPase activity. METHODS AND RESULTS: We examined cTnI phosphorylation in left ventricular myocardium collected from failing hearts at the time of transplant (n=20) and normal hearts from trauma victims (n=24). The relative amounts of actin, tropomyosin, and TnI did not differ between failing and normal myocardium. Using Western blot analysis with a monoclonal antibody (MAb) that recognizes the striated muscle TnI isoforms, we confirmed that the adult human heart expresses only cTnI. A cTnI-specific MAb recognized two bands of cTnI, designated cTnI1 and cTnI2, while a MAb whose epitope is located in the cTnI-specific NH2-terminus recognized only cTnI1. Alkaline phosphatase decreased the relative amount of cTnl1, while protein kinase A and protein kinase C increased cTnI1. The percentage of cTnI made up of cTnI1, the phosphorylated form of TnI, is greater in the normal than the failing human heart (P<.00). CONCLUSIONS: This phosphorylation difference could underlie the reported greater myofibrillar calcium sensitivity of failing myocardium. The functional consequence of this difference may be an adaptive or maladaptive response to the lower and longer calcium concentration transient of the failing heart, eg, enhancing force development or producing ventricular diastolic dysfunction.

High glucose inhibits nitric oxide production in cultured rat mesangial cells.

Hyperglycemia directly contributes to the development of diabetic nephropathy. A high-serum glucose concentration alters intraglomerular hemodynamics and promotes deposition of extracellular matrix in the kidney. Nitric oxide (NO) is a short-lived messenger molecule that participates in the regulation of renal blood flow, GFR, and mesangial matrix accumulation. Therefore, in this study it was tested whether high glucose directly modulates NO synthesis by rat mesangial cells in vitro by measuring the accumulation of nitrite, the stable metabolite of NO, in the incubation media. Raising the external glucose concentration to 33.3 mM for 24 to 72 h reduced nitrite levels in cell supernatants in a time-dependent manner to a nadir of 14 +/- 3% of the amount in normal glucose media (5.6 mM) (P < 0.01). The decline in NO synthesis in high glucose media was paralleled by decreased cyclic guanosine monophosphate generation; however, there was no alteration in rat mesangial cell expression of inducible NO synthase protein. The suppressive effect of high glucose on NO production by mesangial cells was not modified by inhibition of protein kinase C (H-7), the addition of antioxidants (vitamin E or superoxide dismutase), or a pan-specific anti-transforming growth factor-beta antibody. An elevated ambient glucose caused a time-dependent reduction in mesangial cell L-arginine content. Addition of L-arginine (10 to 20 mM) to external media partially reversed the inhibitory effect of high glucose on mesangial cell NO production in a dose-dependent manner. The highest dose of L-arginine (20 mM) increased mesangial cell L-arginine content to comparable levels in normal and high glucose media. These results indicate that high glucose causes depletion of L-arginine in mesangial cells and compromises NO synthesis. Limitation in the metabolic precursor and other, as yet unidentified, factors act to reduce NO production by mesangial cells in the presence of an elevated ambient glucose level, a change that may play a role in the development of diabetic glomerulosclerosis.

HPLC method for monitoring SDZ PSC 833 in whole blood.

P-glycoprotein (Pgp) is a 170-kDa membrane transporter that mediates drug efflux and is an effector of multidrug resistance. SDZ PSC 833 (PSC), a nonimmunosuppressive cyclosporine that potently modulates Pgp, is currently under clinical evaluation in patients with cancer. We have developed a reversed-phase HPLC assay for determining PSC blood concentrations that utilizes a step gradient with linear segments to resolve PSC into two distinct peaks (likely to be keto and enol isomers). To clinically validate the assay, PSC concentrations were obtained by HPLC from nine patients receiving oral doses of 5 mg/kg every 6 h. Values ranged from 0.91 to 5.4 mg/L during the dosing period, comparable with concentrations of PSC that modulate Pgp in vitro. In addition, we investigated the immunoreactivity of the Abbott TDx cyclosporin A (CsA) monoclonal whole-blood assay for PSC. The TDx CsA assay cross-reacts approximately 17% with PSC as determined by adding known amounts of PSC to whole blood. When PSC concentrations obtained by the TDx CsA assay were divided by 0.17, we found agreement between the TDx CsA assay and the HPLC PSC assay for samples from nine patients.

Phosphorylation of calmodulin in the first calcium-binding pocket by myosin light chain kinase.

In smooth muscle and specific nonmuscle cells the phosphorylation of the regulatory myosin light chains by myosin light chain kinase (MLCK) is an obligatory step in actin-induced activation of myosin ATPase and subsequent contractile events. We have previously demonstrated that CaM phosphorylated by casein kinase II fails to activate bovine platelet MLCK (Sacks et al. (1992) Biochem. J. 283, 21-24). While myosin light chains are perceived as the only known substrate for MLCK phosphorylation activity, we now show that MLCK phosphorylates CaM. This phosphorylation of CaM is dependent upon the presence of basic peptides such as poly-L-arginine (optimal basic peptide/CaM ratio = 0.08) and is stimulated by saturating [Ca2+] (K0.5 = 16 microM). CaM phosphorylation was inhibited by KT5926, a specific MLCK inhibitor, with a dose-dependency identical to that for inhibition of myosin light chain phosphorylation. Native and MLCK-phosphorylated CaM were indistinguishable in activating MLCK to phosphorylate myosin light chains. Interestingly, MLCK in which the CaM-binding site has been removed is able to phosphorylate CaM in a Ca(2+)-independent manner, suggesting that two CaM molecules bind to intact MLCK simultaneously, one on the inhibitory (pseudosubstrate) domain and one at the catalytic site. CaM phosphorylation by MLCK occurred exclusively on Thr 29 (90%) and Thr 26 (10%) in the first Ca(2+)-binding pocket. In summary, CaM phosphorylation by MLCK differs from CaM phosphorylation catalyzed by other kinases (i.e., the insulin receptor or casein kinase II) in both basic peptide and Ca2+ requirements as well as in the sites of phosphorylation. Further investigations of this model may provide insight into the mechanisms of MLCK activation and substrate recognition.

Identification of insulin-stimulated phosphorylation sites on calmodulin.

Insulin enhances calmodulin phosphorylation in vivo. To determine the insulin-sensitive phosphorylation sites, phosphocalmodulin was immunoprecipitated from Chinese hamster ovary cells expressing human insulin receptors (CHO/IR). Calmodulin was constitutively phosphorylated on serine, threonine, and tyrosine residues, and insulin enhanced phosphate incorporation on serine and tyrosine residues. Phosphocalmodulin immunoprecipitated from control and insulin-treated CHO/IR cells, and calmodulin phosphorylated in vitro by the insulin receptor kinase and casein kinase II were resolved by two-dimensional phosphopeptide mapping. Several common phosphopeptides were detected. The phosphopeptides from the in vitro maps were eluted and phosphoamino acid analysis, manual sequencing, strong cation exchange chromatography, and additional proteolysis were performed. This strategy demonstrated that Tyr-99 and Tyr-138 were phosphorylated in vitro by the insulin receptor kinase and Thr-79, Ser-81, Ser-101 and Thr-117 were phosphorylated by casein kinase II. In vivo phosphorylation sites were identified by comigration of phosphopeptides on two-dimensional maps with phosphopeptides derived from calmodulin phosphorylated in vitro and by phosphoamino acid analysis. This approach revealed that Tyr-99 and Tyr-138 of calmodulin were phosphorylated in CHO/IR cells in response to insulin. Additional sites remain to be identified. The identification of the insulin-stimulated in vivo tyrosine phosphorylation sites should facilitate the elucidation of the physiological role of phosphocal-modulin.

Facile, in situ matrix-assisted laser desorption ionization-mass spectrometry analysis and assignment of disulfide pairings in heteropeptide molecules.

During a routine analysis of disulfide-linked synthetic heterodipeptides by matrix-assisted laser desorption ionization (MALDI) mass spectrometry with linear detection we observed not only the expected mass of the dipeptide, but also the mass of the individual constituent monomer peptides. This was surprising because the peptide was purified as an intact dipeptide and no overt attempt was made to reduce the disulfide linkage before mass analysis. In contrast, analysis of the same sample by electrospray ionization mass spectrometry gave the mass of the dipeptide only. To investigate this further, two additional model heterodipeptides were prepared and all three were used to systematically study several matrix-assisted laser desorption ionization mass spectrometry parameters. These parameters were three different matrices (alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, and sinapinic acid), both positive and negative modes of detection, and varying the acceleration voltage from 5 to 20 kV. Except for the sinapinic acid matrix where poor-quality spectra were obtained, all three model heterodipeptides fragmented under the tested conditions in a manner consistent with the cleavage of disulfide bonds, although the absolute level was sample dependent. The precise mechanism of disulfide cleavage during analysis is unknown, but the cleavage we observed appears to originate during the initial ionization event. Because the MALDI process involves irradiating samples with a laser, the fragmentation of disulfide-linked peptides that we observe bears some resemblance to light-induced homolytic cleavage of aqueous solutions of the amino acid cystine, although other mechanisms for fragmentation are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural stability of short subsequences of the tropomyosin chain.

The native tropomyosin molecule is a parallel, registered, alpha-helical coiled coil made from two 284-residue chains. Long excised subsequences (> or = 95 residues) form the same structure with comparable thermal stability. Here, we investigate local stability using shorter subsequences (20-50 residues) that are chemically synthesized or excised from various regions along the protein chain. Thermal unfolding studies of such shorter peptides by CD in the same solvent medium used in extant studies of the parent protein indicate very low helix content, almost no coiled-coil formation, and high thermal lability of such secondary structure as does form. This behavior is in stark contrast to extant data on leucine-zipper peptides and short "designed" synthetic peptides, many of which have high alpha-helix content and form highly stable coiled coils. The existence of short coiled coils calls into question the older idea that short subsequences of a protein have little structure. The present study supports the older view, at least in its application to tropomyosin. The intrinsic local alpha-helical propensity and helix-helix interaction in this prototypical alpha-helical protein is sufficiently weak as to require not only dimerization, but macro-molecular amplification in order to attain its native conformation in common benign media near neutral pH.

Tyrosine-phosphorylated calmodulin has reduced biological activity.

Calmodulin is phosphorylated by the purified insulin receptor on tyrosine residues with a maximum stoichiometry of 1 mol phosphate/mol of calmodulin. Isolated tryptic phosphopeptides were sequenced by manual Edman degradation and demonstrated that calmodulin is equally phosphorylated on tyrosine 99 and tyrosine 138. Phosphorylated calmodulin has a decreased affinity (K0.5 = 4.2 nM) for the 63-kDa isozyme of cyclic nucleotide phosphodiesterase compared to nonphosphorylated calmodulin (K0.5 = 2.1 nM). The K0.5 for Ca2+ is marginally increased from 2.8 to 3.2 microM in the presence of phosphotyrosyl calmodulin. The effect of the calmodulin antagonist, mastoparan, was investigated to determine whether mastoparan would differentially inhibit calmodulin- or phosphocalmodulin-dependent enzyme activity. The IC50 of mastoparan is fourfold lower for phosphotyrosyl calmodulin compared to nonphosphorylated calmodulin. Phosphorylation of calmodulin may provide a mechanism for the differential regulation of calmodulin-dependent enzymes. These observations further support a potentially important regulatory function of calmodulin phosphorylation in signal transduction.

Disulfide bonds required to assemble functional von Willebrand factor multimers.

The hemostatic functions of human von Willebrand Factor (vWF) depend on the normal assembly of disulfide-linked multimers from approximately 250-kDa subunits. Subunits initially form dimers through disulfide bonds near the COOH terminus. Dimers then form multimers through disulfide bonds near the NH2 terminus of each subunit. Previous studies of plasma vWF and recombinant vWF fragments indicate that 1 or more of the Cys residues at position 459, 462, and 464 form intersubunit disulfide bonds. No evidence has been reported that vWF multimer formation involves additional intersubunit bonds. To probe the disulfide bond requirements for multimer formation, mutant vWF proteins were expressed in which all 3 Cys residues at positions 459, 462, and 464 were changed to either Gly or Ala. Surprisingly, none of these cysteines appears to be necessary for efficient multimer assembly. Furthermore, recombinant vWF with Gly or Ala at all three positions induces platelet aggregation in the presence of ristocetin and binds to platelet glycoprotein Ib, factor VIII, and collagen in a manner similar to wild-type recombinant vWF. These results suggest that other intersubunit disulfide bonds must exist. Direct evidence for such a bond was obtained by characterization of tryptic fragments of vWF. By Edman degradation, amino acid composition, and mass spectrometry, a disulfide bond was demonstrated between Cys379 residues of adjacent vWF subunits. Thus, intersubunit disulfide bonds involving Cys379 and 1 or more of the Cys residues at positions 459, 462, and 464 connect the NH2-terminal ends of the vWF subunits in a parallel orientation.

Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase II.

IRS-1, a principal substrate of the insulin receptor, is phosphorylated on serine, threonine, and tyrosine residues in a variety of tissues during insulin stimulation. Casein kinase II, an insulin-sensitive serine/threonine kinase, catalyzed the in vitro incorporation of 1 to 2 mol of phosphate/mol of recombinant rat IRS-1. Two-dimensional phosphopeptide mapping of IRS-1 phosphorylated by casein kinase II in vitro and IRS-1 immunoprecipitated from intact Chinese hamster ovary cells demonstrated multiple common phosphopeptides, suggesting that overexpressed IRS-1 is a substrate for casein kinase II in these cells. Moreover, the common phosphopeptides that appeared to be insulin-sensitive in intact cells comprised 22% of casein kinase II-catalyzed 32P incorporation into IRS-1 in vitro. These data suggest that casein kinase II mediates a portion of the insulin-stimulated serine/threonine phosphorylation of overexpressed IRS-1 in vivo. By using phosphoamino acid analysis, strong cation exchange analysis, manual Edman degradation, and automated amino acid sequencing, Thr-502 was identified as the major casein kinase II-catalyzed phosphorylation site in rat IRS-1. Furthermore, Ser-99, an additional site labeled at low yield, appeared to be contained in an insulin-sensitive phosphopeptide. Thus, casein kinase II-catalyzed phosphorylation of IRS-1 may be a component of the intracellular insulin signalling cascade.

Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests.

Several published procedures have been combined to develop a general strategy for the specific identification and isolation of the acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation of the substrate to block lysine NH2 groups; (ii) enzymatic digestion of the modified protein; (iii) automated phenylisothiocyanate derivatization of the protease derived fragments to block newly generated "free" N-termini; and (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed-phase retention time and a greater UV (210-297 nm) profile compared to the corresponding control (-phenylisothiocyanate) digest. The N-terminal acetylated fragment shows neither a retention time shift nor an augmented UV profile. To validate each process step, synthetic peptides and acetylated-N-terminal proteins of known sequence were used as test samples. The desired fragment was isolated from three proteins and positively identified by electrospray mass spectrometry and amino acid composition. Proteins with other N-terminal blocking groups should be amenable to this procedure.

Casein kinase II-catalysed phosphorylation of calmodulin is altered by amino acid deletions in the central helix of calmodulin.

Calmodulin is phosphorylated by casein kinase II on Thr-79, Ser-81, Ser-101 and Thr-117. To determine the consensus sequences for casein kinase II in intact calmodulin, we examined casein kinase II-mediated phosphorylation of engineered calmodulins with 1-4 deletions in the central helical region (positions 81-84). Total casein kinase II-catalyzed phosphate incorporation into all deleted calmodulins was similar to control calmodulin. Neither CaM delta 84 (Glu-84 deleted) nor CaM delta 81-84 (Ser-81 to Glu-84 deleted) has phosphate incorporated into Thr-79 or Ser-81, but both exhibit increased phosphorylation of residues Ser-101 and Thr-117. These data suggest that phosphoserine in the +2 position may be a specificity determinant for casein kinase II in intact proteins and/or secondary structures are important in substrate recognition by casein kinase II.

Insulin-stimulated phosphorylation of calmodulin.

Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling.