Prospective assessment of serum periostin as a biomarker for diagnosis and monitoring of eosinophilic oesophagitis.

BACKGROUND: Periostin is highly expressed in eosinophilic oesophagitis (EoE), but has not been extensively studied as a non-invasive biomarker. AIM: To assess whether serum periostin distinguished EoE from controls at baseline, had utility for monitoring treatment response, or was associated with IL-13 levels. METHODS: This was a sub-analysis of a prospective cohort study of adults undergoing out-patient upper endoscopy. Incident cases of EoE were diagnosed per consensus guidelines. Controls were subjects with either GERD or dysphagia without EoE. EoE patients were treated with swallowed/topical steroids and had repeat endoscopy/biopsy. Serum periostin levels for cases and controls were compared at baseline, and pre/post-treatment levels were compared for cases. Serum IL-13 and tissue expression of periostin were also assessed. RESULTS: A total of 61 incident EoE cases and 87 controls were analysed. Despite a marked increase in tissue periostin expression in cases, the median baseline serum periostin level was only slightly higher in cases than controls (22.1 ng/mL vs. 20.7; P = 0.04); there was no change in post-treatment levels. There was also no difference in serum periostin for cases by histologic response or atopic status. There was a strong trend towards higher serum IL-13 levels in cases in the highest periostin quartile (57.1 pg/mL vs. 2.6; P = 0.07). CONCLUSIONS: Serum periostin levels were similar in cases and controls, and there were no changes post-treatment. Given elevated IL-13 levels in the EoE patients with the highest periostin levels, future studies could explore periostin as a biomarker in EoE, perhaps in the setting of anti-IL-13 therapy.

Ozanimod (RPC1063) is a potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) agonist with autoimmune disease-modifying activity.

BACKGROUND AND PURPOSE: Sphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya™). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases. EXPERIMENTAL APPROACH: The specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4(+) CD45RB(hi) T cell adoptive transfer colitis) was assessed. KEY RESULTS: RPC1063 was specific for S1P1 and S1P5 receptors, induced S1P1 receptor internalization and induced a reversible reduction in circulating B and CCR7(+) T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models. CONCLUSIONS AND IMPLICATIONS: S1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic.

CD80 costimulation is required for Th2 cell cytokine production but not for antigen-specific accumulation and migration into the lung.

The CD28 ligands CD80 and CD86 are expressed on APC, and both provide costimulatory function. However, the reason for the expression of two separate CD28 ligands remains unclear. We have previously shown that blockade of CD80 costimulation by Y100F-Ig, a CTL-associated Ag-4 (CTLA4)-Ig mutant that does not bind CD86, inhibits the development of lung inflammatory immune responses, but does not affect blood eosinophilia or Ab production. Each of those responses was inhibited by treatment with CTLA4-Ig, which binds both CD80 and CD86. To clarify the mechanism underlying these observations we have developed a model of lung inflammation using adoptively transferred CD4(+) T cells expressing a Valpha11(+)Vbeta3(+) transgenic TCR specific for I-E(k) and moth cytochrome c. Treatment with Y100F-Ig inhibited the induction of lung eosinophilia in adoptively transferred mice. However, Y100F-Ig did not detectably affect the accumulation of Ag-specific T cells at the site of peptide deposit or in the draining lymphoid tissues. Acquisition of an activated phenotype and expression of adhesion molecules required for migration into the lung were modestly affected. Importantly, treatment with Y100F-Ig diminished the ability of T cells to produce the cytokines IL-4 and IL-5 following intranasal challenge with Ag. All the responses examined were severely inhibited by treatment with CTLA4-Ig. We conclude that T cells require CD80 costimulation for the optimal production of IL-5 following intranasal administration of Ag. Decreased IL-5 production is the most likely explanation for the diminished airway eosinophilia observed.

Crystallization and preliminary X-ray analysis of CTLA-4 (CD152) membrane-external domain.

CTLA-4 (CD152) is involved in T-lymphocyte co-stimulatory pathways modulating both humoral and cellular immune response. The membrane-external domain has been prepared and crystallized. The unit-cell parameters are a = b = 43, c = 143 A with the symmetry of space group P3(1)21 or its enantiomer and the crystals diffract to 2. 7 A resolution at synchrotron beamlines.

Differential requirement for CD80 and CD80/CD86-dependent costimulation in the lung immune response to an influenza virus infection.

The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-gamma production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-gamma by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86.

CTLA4-Ig inhibits optimal T helper 2 cell development but not protective immunity or memory response to Nippostrongylus brasiliensis.

The B7 co-stimulatory pathway is critical to T cell activation, however its role in the generation of Th2 cells in vivo remains controversial. We have studied the role of B7 co-stimulation in the development of a Th2 immune response to the nematode parasite Nippostrongylus brasiliensis. Blockade of B7 co-stimulation with murine CTLA4-Ig (mCTLA4-Ig) resulted in decreased Th2 cell development as determined by IL-4 and IL-5 cytokine production in vitro. It also resulted in lowered Th2 cell effector function in vivo, with marked reductions in IgE production. Blood eosinophilia was variably affected by mCTLA4-Ig treatment, which resulted in both slight and very severe inhibition in different experiments. However, an effective immune response was still evident as demonstrated by the further reduction of cytokine production, IgE titers, and blood eosinophilia in mice treated with a combination of mCTLA4-Ig and anti-CD4 mAb, and by the ability of mCTLA4-Ig-treated mice to expel adult worms. In addition, mCTLA4-Ig treatment did not alter the development of a memory response following secondary infection with N. brasiliensis, with the exception of IgE production. We conclude from these results that B7 co-stimulation is required in this experimental model for optimal Th2 cell development and effector function in vivo but is not necessary for protective immunity.

Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.

The structure of human CTLA-4 reveals that residues Met 99, Tyr 100 and Tyr 104 of the M99YPPPY104 motif are adjacent to a patch of charged surface residues on the A'GFCC' face of the protein. Mutation of these residues, which are conserved in the CTLA-4/CD28 family, significantly reduces binding to CD80 and/or CD86, implicating this patch as a ligand binding site.

Both extracellular immunoglobin-like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28.

The B7-related molecules CD80 and CD86 are expressed on antigen-presenting cells, bind the homologous T cell receptors CD28 and CTLA-4, and trigger costimulatory signals important for optimal T cell activation. All four molecules are immunoglobulin superfamily members, each comprising an extracellular Ig variable-like (IgV) domain, with CD80 and CD86 containing an additional Ig constant-like (IgC) domain. Despite limited sequence identity, CD80 and CD86 share similar overall receptor binding properties and effector functions. We have identified, by site-directed mutagenesis of soluble forms of CD80 and CD86, residues in both the IgV and IgC domains that are important for CTLA4Ig and CD28Ig binding. Mutagenesis in the IgV domain of CD80 identified 11 amino acids that support receptor binding. Many of these residues are conserved in the B7 family, are hydrophobic, and approximately map to the GFCC'C" beta-sheet face of an IgV fold. Mutagenesis of corresponding residues in CD86 established that some, but not all, of these residues also played a role in CD86 receptor binding. In general, mutations had a similar effect on CTLA4Ig and CD28Ig binding, thereby indicating that both receptors bind to overlapping sites on CD80 and CD86. Further, mutagenesis of several conserved residues in the ABED beta-sheet face of the IgC domain of CD80 completely ablated receptor binding. Point mutagenesis had a more pronounced effect than complete truncation of the IgC domain. Thus, full CTLA4Ig and CD28Ig binding to B7 molecules is dependent upon residues in the GFC'C" face of the IgV domain and the ABED face of the IgC domain.

Endoproteolytic processing of recombinant proalbumin variants by the yeast Kex2 protease.

The yeast Kex2 protease is regarded as the prototype of the eukaryotic family of subtilisin-like serine proteases involved in processing after dibasic amino acid sequences. Here we investigate the specificity of Kex2 using recombinant human proalbumin variants. Proalbumins with the processing site sequences Arg-Arg and Lys-Arg were cleaved after the dibasic sequence at approximately the same rate by Kex2 in vitro, and yeast expressing either of these sequences secreted mature albumin into the culture medium. As expected, the Arg-Gly-Val-Phe-His-Arg-albumin (proalbumin Lille) was not a substrate for Kex2 and neither was the Arg-Gly-Arg-Phe-His-Arg-albumin. In contrast to the mammalian endoproteases furin and the hepatic proalbumin convertase, the Kex2 protease was adversely affected by a P4 arginine. There was an 85% decrease in the cleavage of Arg-Gly-Arg-Phe-Arg-Arg-albumin compared with normal; also chicken proalbumin with an Arg-Phe-Ala-Arg processing site sequence was not a substrate for Kex2. A P1' arginine had a marked negative effect on processing and N-terminal sequence analysis confirmed that cleavage was occurring at the P1-P1' bond. The sequence context surrounding the classical dibasic site is critical in determining susceptibility to cleavage by the Kex2 protease.

Complementarity determining region 1 (CDR1)- and CDR3-analogous regions in CTLA-4 and CD28 determine the binding to B7-1.

T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.

Immunoglobulin fold characteristics of B7-1 (CD80) and B7-2 (CD86).

B7-1 and B7-2 are expressed on antigen-presenting cells and bind to the CD28 and CTLA-4 receptors on T cells. These interactions trigger a costimulatory pathway that is essential for T-cell activation. B7-1 and B7-2 are members of the immunoglobulin superfamily (IgSF) and, despite sharing common function, have only limited sequence similarity. The B7-1 extracellular region was previously subdivided into 2 IgSF domains, an N-terminal V(ariable)-like domain, followed by a C(onstant)-like domain. We recently reported that the V-like domains of B7-1 and B7-2 share some significant sequence similarities with 3 major histocompatibility complex (MHC)-encoded members of the IgSF. We have now applied inverse folding methodology to assess the compatibility of the B7-1 and B7-2 extracellular region sequences with currently available 3-dimensional structures. In these calculations, the sequences of the N-terminal (V-like) domains in B7-1 and B7-2 were not compatible with known structures, including the IgSF V-set. In contrast, the sequences of the C-like domains were compatible with IgSF C-set structures and were best recognized by the beta 2-microglobulin (beta 2m) domain of MHC Class I. A sequence comparison of the C-like domains in the B7 molecules showed that 11 of 17 rigorously conserved residues in B7-1 and B7-2 are not IgSF C-1 set consensus residues. When mapped onto the corresponding positions of the beta 2m structure, the conserved residues in B7 cluster on the surface, where they may interact with the B7 V-like domain or other molecules.

Identification of hyaluronic acid binding sites in the extracellular domain of CD44.

CD44 is a polymorphic glycoprotein expressed on the surface of many tissues and cell lines which has been implicated in a number of cellular functions including lymphocyte homing to mucosal lymphoid tissue (Peyers patches), leukocyte activation, lymphopoiesis, and tumor metastasis. The predominant isoform found on human leukocytes, CD44H, is a receptor for hyaluronic acid. Because of the prominent role CD44 plays in diverse biological processes, we set out to identify the hyaluronic acid binding site(s) in the extracellular domain of CD44H. Using truncation and site-directed mutagenesis we identified two regions containing clusters of conserved basic residues which are important in hyaluronic acid binding. One of these regions is situated near the NH2 terminus and is homologous to other hyaluronic acid binding proteins including cartilage link protein. The other more membrane proximal region lies outside the link protein homologous domain. Mutagenesis of basic residues within these regions established their role as determinants in hyaluronic acid binding. Mutation of Arg 41, a position where a basic residue is conserved in all hyaluronic acid binding proteins, completely abolished binding suggesting that this residue plays a critical role in hyaluronic acid binding.

Albumin Rugby Park: a truncated albumin variant caused by a G-->C splice-site mutation in intron 13.

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.

Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high-performance anion-exchange chromatography and nuclear magnetic resonance spectroscopy.

The characterisation of oligosaccharides present on albumin Casebrook, a glycoprotein variant of human serum albumin, which contains an N-linked oligosaccharide at an attachment site formed by a point mutation of 494 Asp-->Asn, is described. The monosaccharide compositional analysis of purified glycopeptides suggested the presence of complex biantennary carbohydrate structures. The oligosaccharides which were released by N-glycosidase-F appeared to be a single molecular species according to their retention on high-performance anion-exchange chromatography. The structure of the oligosaccharide was suggested by sequential exoglycosidase digestions and confirmed by proton nuclear magnetic resonance spectroscopy. It was concluded that the oligosaccharides were essentially homogeneous and consisted of an alpha(2-6)-desialylated complex biantennary glycan.

The processing of human proinsulin and chicken proalbumin by rat hepatic vesicles suggests a convertase specific for X-Y-Arg-Arg or Arg-X-Y-Arg sequences.

Vesicles from rat and chicken livers contain very similar Ca2(+)-dependent proteases that respectively cleave (human) proalbumin at an Arg-Arg site and chicken proalbumin at an Arg-Phe-Ala-Arg site. Similar Ca2(+)-dependent proteases are also present in pancreatic secretory granules and cleave proinsulin at two sites, Arg-Arg and Lys-Arg. The mammalian liver processes a large variety of different proproteins and in order to assess its processing site requirements, we investigated the ability of rat hepatic vesicle extracts to cleave purified chicken proalbumin and human proinsulin. Despite having only a monobasic processing site, chicken proalbumin was cleaved faster than human proalbumin which not only contains a dibasic site, but has an identical propeptide to that of the rat's own proalbumin. Human proinsulin was processed by the rat liver extracts; however, no mature insulin was produced. Cleavage occurred in only one place, presumably the Arg-Arg site at the B-C chain junction. This suggests that the mammalian liver might not contain a Type II Lys-Arg-directed convertase, only a Type I Arg-Arg-specific enzyme. The Type I enzyme that cleaves human proalbumin appears to be the same activity that cleaves chicken proalbumin, suggesting a specificity for either X-Y-Arg-Arg or Arg-X-Y-Arg sequences. This proposal is in keeping with the processing site motif of some 16 different proproteins that are known to be processed in the liver and is entirely consistent with the known in vivo specificity of the enzyme defined by naturally occurring variants of human proproteins.

Prediction of the site of signal peptidase cleavage in normal and variant human preproalbumin.

We describe the use of a template for the recognition site of signal peptidase to give score predictions for cleavage at normal and aberrant sites in human and chicken preproalbumin. As expected, the highest score obtained for human preproalbumin was at the normal cleavage site between -7 Ser and -6 Arg. The mutation in preproalbumin Kaikoura (-2 Arg----Cys) introduced an aberrant cleavage site in the propeptide between the new -2 Cys and -1 Arg. Although the new site had a lower template score (6.88) than the normal site (9.99), it is cleaved in vivo about five times faster. The presence of minor proportions of des-Asp and des-Asp-Ala-albumin in normal human plasma was not found to correlate with possible aberrant signal peptidase cleavage. These truncated albumin species, therefore, appear to be generated through cleavage of mature albumin in circulation. Chicken preproalbumin had only one probably signal peptidase cleavage site, the one utilised in vivo. Template scores should be regarded as qualitative predictions rather than definitive quantitative indicators.

Structural characterization of a glycoprotein variant of human serum albumin: albumin Casebrook (494 Asp----Asn).

Albumin Casebrook is an electrophoretically slow genetic variant of human albumin with a relative molecular mass 2.5 kDa higher than normal albumin. It constitutes about 35% of total serum albumin in heterozygous carriers. The decrease in negative charge observed on incubation with sialidase suggested the presence of a carbohydrate moiety and the normalization of molecular weight following treatment with Endo-F indicated that this was an N-linked oligosaccharide. Partial acid hydrolysis and limited tryptic digestion established that the oligosaccharide was located in the C-terminal domain, between residues 367 and 585. Tryptic, chymotryptic and S. aureus V8 proteinase digestions were carried out and the resulting glycopeptides were purified on concanavalin A-Sepharose. Peptide mapping of bound and unbound fractions followed by amino acid composition and sequence analysis, established a point mutation of 494 Asp----Asn. This introduces an Asn-Glu-Thr N-linked oligosaccharide attachment sequence centered on Asn-494 and explains the increase in molecular mass. There was no apparent pathology associated with the presence of this new glycosylated albumin, which was detected in two unrelated individuals of Anglo-Saxon descent.

Rapid detection and initial characterization of genetic variants of human serum albumin.

We have studied the detection and classification of genetic variants of human serum albumin by electrophoresis. Samples from 10 patients who were heterozygous for eight different albumin variants were studied by two methods. In agarose gel electrophoresis, each of these variants has an abnormal mobility and can be classified on the basis that structural changes at the N-terminus abolish 63Ni binding. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole serum, glycosylated variants are easily detected because of their greater apparent molecular mass.

Specificity of yeast KEX2 protease for variant human proalbumins is identical to the in vivo specificity of the hepatic proalbumin convertase.

Yeast KEX2 protease was examined as a potential model for a human proprotein convertase and, in all respects, mimicked the predicted properties of a proalbumin convertase. The enzyme rapidly cleaved the propeptide Arg-Gly-Val-Phe-Arg-Arg from the NH2-terminal end of proalbumin but, unlike trypsin, failed to cleave physiologically unprocessed human proalbumin variants. There was little or no cleavage of proalbumin Lille (Arg-2----His) or Christchurch (Arg-1----Gln), and there was negligible cleavage of proalbumin Blenheim (Asp1----Val), despite the fact that it retains the dibasic processing signal. Proalbumin Kaikoura (Arg-2----Cys), which appears to be partially processed in vivo, was cleaved at about half the rate of normal proalbumin despite the absence of a diarginyl sequence. Restoration of a dibasic site through aminoethylation of the new cysteine increased the rate of cleavage to near that of normal proalbumin. The KEX2-catalyzed cleavage of normal proalbumin was found to be independent of pH between pH 6.0 and 8.0. Antitrypsin Pittsburgh (Met358----Arg), a predicted specific inhibitor of in vivo proalbumin cleavage, inhibited KEX2 in a reversible manner. A molar excess of thrombin over antitrypsin Pittsburgh relieved the inhibition of KEX2, suggesting that a covalent complex is not formed between KEX2 and the inhibitor.

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.