Location of crosslinks in chemically stabilized horseradish peroxidase: implications for design of crosslinks.

The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker.     

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.     

Protein HMG-17 is hyper-expressed in rat glucagonoma. Single-step isolation and sequencing

High-mobility-group protein 17 (HMG-17) was identified by reversed-phase high-performance liquid chromatography analysis as a major component in acidic extracts of transplantable rat glucagonoma tissue but not in insulinoma tissue of similar origin. The peptide was purified in a single step and the entire sequence of 89 amino acids was determined. Rat HMG-17 has a molecular mass of 9238 Da and shows strong similarity to human, bovine (94.4%) and chicken (88.8%) HMG-17. Six of the seven residues which vary among the mammalian sequences are located within a short segment (positions 64-83) present in the acidic, non-DNA-binding C-terminal part of HMG-17. This region shows least similarity to the otherwise related proteins HMG-14 and H6 (a trout HMG protein). Interestingly, four of the six variable positions are Asp in rat HMG-17 which results in an overall net increase in the negative charge of the C-terminal region. The nature of selective hyper-expression of HMG-17 in glucagon but not in insulin-producing tumor tissue remains to be clarified.     

Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen

The Tn antigen (GalNAc alpha-O-Ser/Thr) as defined by the binding of the lectin, helix pomatia agglutinin (HPA) or anti-Tn monoclonal antibodies, is known to be exposed in a majority of cancers, and it has also been shown to correlate positively with the metastatic capacity in breast carcinoma. The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1), which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin-embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4) did to some extent overlap with the presence of IgA1 in the tumours, but differences were seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence of Tn positive IgA in serum. On average 51% of the tumour cells in the individual breast cancer tumour sections showed staining for IgA1. The overall amount of staining in the invasive part of the tumour with the anti Tn antibody was 67%, and 93% with HPA. The intra-expression or uptake of IgA1 in breast cancer makes it a new potential carrier of the tumour associated and immunogenic Tn antigen.     

Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4

Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9.9+/-0.2 by spectrophotometric titration, the fast phase amplitude, and decrease in the apparent second order rate constant, respectively. An acidic pK(a) at 3.2+/-0.3 was also determined from the apparent second order rate constant. The reactions of soybean peroxidase compounds I and II with veratryl alcohol at pH 2.44 give very similar second order rate constants, k(2)=(2.5+/-0.1)x10(4) M(-1) s(-1) and k(3)=(2.2+/-0.1)x10(4) M(-1) s(-1), respectively, which is unusual. The electronic absorption spectra of compounds I, II and III at pH 7.07 show characteristic bands at 400 and 651 nm (compound I), 416, 527 and 555 nm (compound II), and 414, 541 and 576 nm (compound III). No additional intermediates were observed.     

A Protein Deep Sequencing Evaluation of Metastatic Melanoma Tissues

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma “genomic subtypes”, (“pigmentation” and “high immune”) revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.     

The amyloplast proteome of potato tuber

Potato (Solanum tuberosum) is the fourth largest crop worldwide in yield, and cv. Kuras is the major starch potato of northern Europe. Storage starch is packed densely in tuber amyloplasts, which become starch granules. Amyloplasts of soil-grown mini-tubers and agar-grown micro-tubers of cv. Kuras were purified. The mini-tuber amyloplast preparation was enriched 10-20-fold and the micro-tuber amyloplast approximately fivefold over comparative total protein extracts. Proteins separated by SDS-PAGE were digested with trypsin, analysed by mass spectrometry and identified by mascot software searches against an in-house potato protein database and the NCBI non-redundant plant database. The differential growth conditions for mini- and micro-tubers gave rise to rather different protein profiles, but the major starch granule-bound proteins were identical for both and dominated by granule-bound starch synthase I, starch synthase II and alpha-glucan water dikinase. Soluble proteins were dominated by starch phosphorylase L-1, other large proteins of the classes 'starch and sucrose metabolism', 'pentose phosphate pathway', 'glycolysis', 'amino acid metabolism', and other proteins such as plastid chaperonins. The majority of the identified proteins had a predicted plastid transit peptide, supporting their presence in the amyloplast. However, several highly expressed proteins had no transit peptide, such as starch phosphorylase H, or had a predicted mitochondrial location. Intriguingly, all polyphenol oxidases, a family of enolases, one transketolase, sulfite reductase, deoxynucleoside kinase-like and dihydroxy-acid dehydrase had twin-arginine translocation motifs, and a homologue to dihydrolipoamide dehydrogenase had a Sec (secretory) motif; these motifs usually target thylakoid-like structures.