Graham J. Hughes

Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA

MDCK cells expressing the polymeric immunoglobulin (poly-Ig) receptor, cocultured with IgA-producing hybridoma cells, transported dimeric IgA (dIgA) from the basolateral into the lumenal compartment, where it was recovered as secretory component-dIgA complexes. The tail of the receptor was phosphorylated on serines 664 and 726. Each serine was mutated to alanine. Appearance of A726 receptor at the basolateral surface was reduced approximately 5-fold. This was accompanied by a approximately 5-fold reduction in dIgA transcytosis. Basolateral delivery of receptor was not affected by mutation A664, and in the absence of dIgA, the receptor accumulated in recycling basolateral endosomes. In coculture, however, dIgA transcytosis by A664 receptor was normal. Thus, entry of receptor into the transcytotic pathway requires Ser-664 phosphorylation only in the absence of dIgA.

Human α and β parvalbumins

alpha and beta parvalbumins are Ca(2+)-binding proteins of the EF-hand type. We determined the protein sequence of human brain alpha parvalbumin by mass spectrometry and cloned human beta parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. beta parvalbumin differs in 54 positions from alpha parvalbumin and lacks the C-terminal amino acid 109. From MS analyses of alpha and beta parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. alpha and beta parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase-chain-reaction techniques. Whereas alpha parvalbumin was found in a number of adult human tissues, beta parvalbumin was restricted to preterm placenta. The pattern of alpha parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, alpha parvalbumin was found in extrafusal and intrafusal skeletal-muscle fibres whereas, in man, alpha parvalbumin was restricted to the muscle spindles. Different functions for alpha and beta parvalbumins are discussed.

Comparison of the high-performance liquid chromatography of peptides and proteins on 100- and 300-Å reversed-phase supports

Abstract The chromatographic separations of peptides and proteins on commercially available 100- and 300-A pore size reversed-phase columns have been compared using various buffer systems. The larger-pore-size packing exhibits a slightly more hydrophilic character while maintaining flow and back-pressure characteristics typical of 10-μm supports. In addition to equal or improved resolving capabilities for smaller amino acid derivatives and peptides, this column material is notably superior to the 100-A for the chromatography of proteins with molecular weights exceeding ca . 15,000.

Formation of peptide impurities in polyester matrices during implant manufacturing

Most peptides are susceptible, in vivo, to proteolytic degradation, and it is difficult to formulate and to deliver them without loss of biological activity. In addition, it is often desirable to release them continuously and at a controlled rate over a period of weeks or months. For these reasons, a controlled release system is suitable. Poly(lactic acid) (PLA) is a biocompatible and biodegradable material that can be used for many applications, including the design of injectable controlled release systems for pharmaceutical agents. Development of these delivery systems presents challenges in the assessment of stability, specially for peptide drugs. By means of an extrusion method, long-acting poly(lactic acid) implants containing vapreotide, a somatostatin analogue, were prepared. The nature of the main degradation product obtained after implant manufacturing was elucidated. It was found that the main peptide impurity was a lactoyl lactyl-vapreotide conjugate. Because lactide are found in small quantities in most commercially available PLA, the influence of residual lactide in the polymeric matrix, on the formation of peptide impurities during manufacturing, was specially investigated. This work demonstrates that the degree of purity of the carrier is of great importance with regard to the formation of peptide impurities.

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma.

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2‐D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N‐terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn‐SOD). No multimeric Mn‐SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn‐SOD are metallo‐enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21 → qter and 6q21‐6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn‐SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.*

A human de‐ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro

Some enzymatic and physicochemical properties of a human ubiquitin‐specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de‐ubiquitinating activity capable of cleaving ‘head‐to‐tail’ polyUb chains as well as isoamide ‘branched’ Ub dimers. K M values show a 10 fold preference for the cleavage of branched Ub dimers over head‐to‐tail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S20,w value of 5.2 S and a molar mass of 110 kDa ± 10%. Because the enzyme cleaves both peptide‐linked and isopeptide‐linked Ub moieties from substrates, we propose to name it de‐ubiquitinase rather than isopeptidase.

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.

Increased stability upon heptamerization of the pore-forming toxin aerolysin

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH2-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180–307 and 401–427, were identified, by limited proteolysis, NH2-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of β structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Elevation of apolipoprotein E in the CSF of cattle affected by BSE

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt‐Jakob disease (CJD) display two unique polypeptide chains by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). In the absence of a well‐defined ante‐mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post‐mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10‐fold increase). These elevated polypeptide chains were identified as apolipoprotein E.

Microsequence analysis: I. Peptide isolation using high-performance liquid chromatography

Peptide mixtures, arising from such diverse sources as tissue extracts, urine or proteolysis, are usually fractionated and characterized by a combination of ion-exchange chromatography, gel-exclusion chromatography and/or electrophoresis. Appropriate combinations of these methods will normally yield a homogeneous product, but their costs, detection limits, analyses times and yields vary. The recently introduced technique of high-performance liquid chromatography is now being adapted to peptideprotein fractionation by numerous laboratories. The great versatility of this technique rests in the various column materials available for normal-phase adsorption (silica), reverse-phase adsorption (hydrocarbon bonded to silica) or ion exchange (ionic groups bonded to silica or polystyrene). To date proteins and large peptide fragments have been separated using both ion exchange (CM-glycophase/CPG [ 1 ] and DEAE-glycophase/CPG [2]) and molecular or steric exclusion supports (glycophase G/CPG [ 1,2]). Smaller peptides resulting from enzymatic hydrolyses [ 1,3-71, solid-phase syntheses [8] and tissue extractions [5,6,9,10] have been chromatographed and/or isolated using reverse phase Cl 8 columns and either isocratic or gradient elution systems. The detection systems, in most cases absorbance, limit both the buffer systems which can be employed and also the sensitivity. These restrictions