Characterization of epitopes of human secretory component on free secretory component, secretory IgA, and membrane-associated secretory component

We have compared the epitopes present in various forms of human secretory component by using a panel of hybridoma-derived antibodies elicited by immunizing mice with free secretory component (FSC) or secretory IgA (sIgA). Enzyme-linked immunosorbent binding assays (ELISA) were used to assess antibody binding to FSC- and SC-containing antigens, including sIgA isolated from milk, reduced and alkylated sIgA, and sIgA assembled in vitro by incubating dimeric IgA with FSC. Immunofluorescence assays were also used to assess binding to a human epithelial tumor cell line (HT29) that expresses secretory component as an integral protein of the plasma membrane. The results can be summarized as follows. 1) Most antibodies from fusions in which sIgA was the immunizing antigen bound preferentially to sIgA. 2) Most antibodies from fusions in which FSC was the immunizing antigen bound preferentially to FSC. 3) Antibodies that bound preferentially to sIgA invariably bound sIgA assembled in vitro; antibodies that bound preferentially to FSC invariably did not. 4) Antibodies that bound readily to both sIgA and FSC were rare in all fusions. 5) The monoclonal antibodies defined at least six classes of epitopes on SC, including epitopes that were a) FSC specific and reduction sensitive, b) FSC specific and reduction insensitive, c) sIgA specific and reduction-sensitive, d) sIgA specific and reduction insensitive, e) shared by FSC and sIgA and reduction-sensitive, and f) shared by FSC and sIgA and reduction-insensitive. 6) Antibodies that mediated intense immunofluorescent staining of secretory component on HT29 cell membranes were rare and constituted a distinct subset of those which recognized epitopes shared by FSC, reduced and alkylated sIgA, and some preparations of native sIgA. Results of these antibody-binding studies indicate that most SC epitopes are not shared by FSC and sIgA. Most SC-related epitopes on sIgA appear to be generated by the physical interaction of SC with dimeric IgA, whereas most epitopes on FSC are masked or altered by this interaction. Finally, epitopes that are shared by membrane SC and FSC and/or sIgA represent a minor and immunochemically distinct subset of epitopes on SC. The high proportion of unique epitopes on the different physical forms of SC suggest that the epitopes of this molecule are highly sensitive to its molecular environment. The monoclonal reagents described here will be useful in studying the structure and function of SC; quantitating FSC, sIgA, and membrane SC; purifying various molecular forms of SC by immunoaffinity chromatography; and localizing SC in human tissues and cultured cells by immunocytochemical techniques.     

Quantification of human urinary free secretory component, secretory and nonsecretory IgA by ELISA

The specific quantification of human urinary free secretory component (FSC), secretory IgA (SIgA) and total IgA using ELISA has been hampered by mutual interferences of these three molecules. Using affinity chromatographically purified antisera an attempt was therefore made to reduce these interferences without necessitating further assay steps. FSC and total IgA were measured in unprocessed urine by means of anti-FSC and anti-IgA as well as alkaline phosphatase-coupled anti-FSC or anti-IgA antisera. SIgA was determined using anti-IgA as well as alkaline phosphatase-coupled anti-FSC. Nonsecretory urinary IgA was calculated from the measured SIgA and total IgA. The mutual interferences of FSC, SIgA or nonsecretory IgA in the three assay systems were low and not relevant for normal samples. Normal urinary concentrations were: FSC 344 +/- (SD) 208 ng/ml (n = 120), SIgA 1,874 +/- 1,133 ng/ml (n = 123) and nonsecretory IgA, depending on the way of standardization, 712 +/- 699 (n = 56) or 878 +/- 732 ng/ml (n = 51). SIgA excretion increased with age. Lower urinary SIgA as well as total and nonsecretory IgA levels were observed in males as compared to females. No correlation evolved between the hormonal status of women and the excretion of FSC, SIgA or IgA. In IgA-deficient patients virtually no nonsecretory IgA or SIgA was detected in the urine while the FSC concentration was in the normal range.     

Improved protein refolding using hollow-fibre membrane dialysis

We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25 degrees C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25 degrees C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield.     

In vitro refolding of PEGylated lipase

Covalent modification of proteins with polyethylene glycol (PEG) has become a well established drug enhancement strategy in the biopharmaceutical industry. The general benefits of PEGylation, such as prolonged serum half-lives or reduced in vivo immunogenicity, are well known. To date, the PEGylation process has been performed with purified proteins, which often requires additional multi-step purification steps to harvest the desired PEGylate. However, it would be beneficial for bioprocessing if 'renaturation,' i.e. in vitro refolding and 'modification,' and PEGylation can be integrated, especially for inclusion body proteins. We investigated the feasibility of protein PEGylation under denaturing conditions and of protein refolding with the attached PEG molecule. Using lipase as a model protein, PEGylation occurred in 8 M urea and covalently attached PEG did not appear to hinder subsequent refolding.     

Determination of the molecular structure of the human free secretory component.

Here, we present the experimental data, leading to determination of the primary structure, the linkage of the carbohydrates and the arrangements of the disulfide bonds of the human free secretory component. Methods of protein chemistry were used. The protein can be divided into five homology regions and is a member of the immunoglobulin superfamily.     

Enhanced in vitro refolding selectivity of the recombinant human insulin-like growth factor I

A systematic study was carried out to optimize production of biologically active recombinant IGF-I with native conformation. Careful optimization of buffer carbonate, pH (9.5), protein concentration (0.5 mg/ml), temperature (25°C), and disulfide-exchange reagents (2 mM reduced glutathione/1 mM oxidized glutathione, 1 mM cysteine, 1 mM -mercaptoethanol, and 2 mM dithiothreitol) allowed a yield of correctly folded recombinant IGF-I as high as 80%, which may be useful for large scale production of IGF-I.This revised version was published online in October 2005 with corrections to the Cover Date.     

Structure of the carbohydrate chain of free secretory component from human milk.

Secretory component from human milk was found to contain 23.4% carbohydrate, which includes galactose, mannose, fucose, glucosamine, and sialic acid. Secretory component could be degraded by pronase or base-borohydride to yield the same, single type of carbohydrate chain. In the glycopeptide produced by pronase digestion, aspartic acid was the only amino acid present in molar quantities after amino acid analysis, which suggests that the carbohydrate moiety is linked to the polypeptide chain at asparagine residues. The positions of links between the various sugar units were studied by methylation analyses of: secretory component, periodate-oxidized and reduced secretory component, the fragment produced by base-borohydride treatment, and the pronase glycopeptide after treatment with specific glycosidases. Sugars released from the glycopeptide by various glycosidases were also quantitated. From the results of these studies a branched chain structure was assigned to the carbohydrate chain of secretory component.     

Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.     

Production and solid-phase refolding of human glucagon-like peptide-1 using recombinant Escherichia coli

A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.     

In vitro protein refolding by chromatographic procedures

In vitro protein refolding is still a bottleneck in both structural biology and in the development of new biopharmaceuticals, especially for commercially important polypeptides that are overexpressed in Escherichia coli. This review focuses on protein refolding methods based on column procedures because recent advances in chromatographic refolding have shown promising results.     

Expression and refolding of recombinant human fibroblast procathepsin D

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.     

The measurement of free nitrendipine in human serum by an equilibrium dialysis - radioreceptor assay

A radioreceptor assay using [³H]nitrendipine and rat cerebral cortical membranes, in conjunction with equilibrium dialysis, measures the unbound (free) level of nitrendipine in human sera. The sensitivity of the assay is 0.1–0.2 picomoles/ml and is linear from 4 × 10^?11 to 4 × 10^?9 M nitrendipine. Other dihydropyridine calcium channel antagonists may be measured using this assay if these compounds are used to generate the standard curve. Blank serum interferes with specific [³H]nitrendipine binding (24 percent inhibition per 20 μ1 serum) whereas serum dialysates do not. Total serum nitrendipine levels may be measured, but the sensitivity of the assay is decreased due to interference by serum. Nitrendipine is highly protein bound in serum (93 – 99 percent). This protein binding is essentially unchanged over a serum concentration from 1 to 100 ng/ml. This assay is suitable for pharmacokinetic and pharmacodynamic studies.     

Probing the topography of free and polymeric Ig-bound human secretory component with monoclonal antibodies.

Secretory component (SC), an integral membrane protein expressed on basolateral surfaces of secretory epithelial cells, mediates the transport of polymeric Ig (PIg) into external secretions. The ectoplasmic segment of SC is released into secretions either in a free form (FSC) or bound to PIg as secretory IgA or IgM. The topography of human SC in its free and PIgA-bound form was studied by using mAb directed against each form of SC. Competition experiments identified a minimum of nine SC epitopes, one of which was dependent on an N-glycosidic moiety. Three of the polypeptide-derived epitopes were displayed on denatured, reduced, and alkylated SC, whereas the others were fully or partially dependent on the native conformation of SC. Epitopes recognized by the latter class of antibodies were mapped to discrete domains of SC, based on amino acid sequence and antibody-binding analysis of limited proteolytic fragments. One of the mAb (6G11), which was directed against an epitope on domain I of SC, inhibited the binding of FSC to PIgA. Overall, our results provide evidence that a region within domain I, as well as protease-sensitive interdomain regions of FSC, become masked or altered when SC binds to PIgA. Furthermore, the binding of SC to PIgA results in conformational changes, or formation of combinatorial epitopes, involving regions within domains II and III of SC but not domain V.     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

When total cytoplasmic RNA from mouse Friend cells is fractionated using oligo(dT)-cellulose or poly(U)-Sepharose chromatography, approximately 20% of the messenger RNA activity (as measured in the reticulocyte lysate cell-free system) remains in the unbound fraction, even though this contains < 0.5% of the poly(A) (as measured by titration with poly(U)). This RNA, operationally defined as poly(A)^?, is found almost entirely in polysome structures in vivo. Its major translation products, as shown by one-dimensional sodium dodecyl sulphate-containing gels, are the histones and actin. Two-dimensional gels (isoelectric focusing: sodium dodecyl sulphate/gel electrophoresis) show that, with the exception of the mRNAs coding for histones, poly(A)^? mRNA encodes similar proteins to poly(A)⁺ mRNA, though in very different abundances. This is directly confirmed by the arrest of the translation of the abundant poly(A)^? mRNAs after hybridization with a complementary DNA transcribed from poly(A)⁺ RNA.RNA sequences which are rare in the poly(A)⁺ RNA are also found in poly(A)^? RNA, as shown by hybridizing a cDNA transcribed from poly(A)⁺ RNA to total and poly(A)^? polysomal RNA. That this does not simply represent a flow-through of poly(A)⁺ RNA is indicated by (i) the lack of poly(A) by hybridizing to poly(U) in this fraction, (ii) the fact that further passage through poly(U)-Sepharose does not remove the hybridizing sequences, (iii) the very different quantitative distribution of proteins encoded by poly(A)⁺ and poly(A)^? RNAs. We also think that it does not result from removal of poly(A) from polyadenylated RNAs during extraction because RNAs prepared using the minimum of manipulations give similar results. The distribution of both total mRNA and α and β globin mRNAs between poly(A)⁺ and poly(A)^? RNA does not change significantly during the dimethyl sulphoxide-induced differentiation of Friend cells.     

Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase

Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.     

On-column refolding of recombinant human interleukin-4 from inclusion bodies

Interleukin-4 (IL4) is a multifunctional cytokine which plays a key role in the immune system. Several antagonists/agonists of IL4 are reported through mutagenesis studies, but their solution structural studies using nuclear magnetic resonance (NMR) spectroscopy are hindered as milligram quantities of isotopically labeled protein are required for structural refinements. In this work, a His-tagged recombinant form of human IL4 was overexpressed in Escherichia coli under the control of a T7 promoter. The resulting inclusion bodies were separated from cellular debris by centrifugation and solubilized by 6M guanidine-HCl in the presence of reducing agents. The denatured IL4 was immobilized on Ni2+-fractogel beads and refolded in a single chromatographic step by gradual removal of denaturant. This protocol yielded 15-20 mg of isotope-enriched protein from 1L of culture grown in minimal medium. The refolded protein was highly pure and was correctly folded as judged by its two-dimensional NMR spectrum. To show the successful application of this refolding protocol to IL4 variants, 15N-labeled Y124D-IL4 was also prepared and its first two-dimensional NMR spectrum was presented.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.     

Comparison of serum free thyroxine measurements by chemiluminescence and equilibrium dialysis

A study of 239 patients compared free thyroxine (FT4) measurements made by equilibrium dialysis (ED) with measurements made using the Magic Lite FT4 chemiluminescence (Cl) immunoassay (Ciba Corning Immunodiagnostics). Patient groups: 41 normals; 27 hyperthyroid; 29 hypothyroid; 37 sick euthyroid; 10 chronic renal failure (CRF) and 25 pregnant patients; 13 oestrogen; 10 heparin; 12 salicylate; and 9 dilantin-treated patients; 3 lipaemic; 5 haemolysed; 6 hyperbilirubinaemic patients; 6 low thyroid binding protein (TBP) and 6 high TBP level patients. The two assays gave comparable results in most groups. Both assays tended to give elevated values in heparinized patients but FT4–ED results were more obviously affected. Pregnant patients and women on oral oestrogen had higher mean values with FT4–ED. In both assays the sick euthyroid and CRF patients had mean FT4 values similar to healthy euthyroid patients; the range of values in sick euthyroid and CRF patients was similar in both assays but wider than in healthy euthyroid patients. A supplemental study of 81 unselected acutely ill patients using FT4–Cl alone confirmed the wider range of values to be anticipated in sick euthyroid patients.