Publications by authors named "Risto Renkonen"

73 Publications

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

Mol Cell Proteomics 2015 Feb 1;14(2):263-76. Epub 2014 Dec 1.

From the ‡Transplantation Laboratory, Haartman Institute, PO Box 21, Haartmaninkatu 3, FI-00014 University of Helsinki, Finland; §HUSLAB, Helsinki University Central Hospital, Helsinki, Finland;

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes.
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http://dx.doi.org/10.1074/mcp.M114.040345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350024PMC
February 2015

Transient elevation of serum 5-HIAA by dietary serotonin and distribution of 5-HIAA in serum protein fractions.

Ann Clin Biochem 2015 Jul 23;52(Pt 4):428-33. Epub 2014 Sep 23.

HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.

Introduction: Dietary serotonin increases urinary secretion of 5-HIAA. A falsely elevated 5-HIAA may lead to incorrect suspicion of a neuroendocrine tumour. Therefore, we determined the effect and duration of dietary serotonin on serum 5-HIAA concentration. We also studied the distribution of 5-HIAA in serum fractions.

Methods: We used serum samples from healthy volunteers (31 women and four men). All test subjects avoided serotonin-containing foods for three days before sample collection. They then ate either pineapple, banana, kiwi fruit, tomato or walnuts and additional blood samples were taken after 2, 4, 6, 24, 48 and 72 h. To study the distribution of 5-HIAA in serum, samples from a healthy individual, a test person who had ingested walnuts, and from a neuroendocrine tumour patient were fractionated by gel filtration chromatography. The fractions were analysed for 5-HIAA.

Results: Serum 5-HIAA concentration increased significantly (P ≤ 0.001) within 2 h after ingestion of serotonin-containing food. After 2 h, 5-HIAA concentration started to decrease and reached the baseline concentration within 24 h. A calculated half-life of 5-HIAA in circulation was 1.3 h. In fractionated serum, 5-HIAA was found not only in free form but also in the albumin and α2-globulin fractions.

Conclusions: The increase of serum 5-HIAA caused by dietary serotonin is significant but transient. Therefore, serotonin-containing foods should be avoided for one day before blood sampling. In serum, 5-HIAA is free and apparently bound to albumin. Minor amounts were also found in the α2-globulin fraction. Our liquid chromatography tandem mass spectrometry assay measures free 5-HIAA in serum.
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http://dx.doi.org/10.1177/0004563214554842DOI Listing
July 2015

Comparison of sialylated N-glycopeptide levels in serum of pancreatic cancer patients, acute pancreatitis patients, and healthy controls.

Proteomics 2014 Aug;14(15):1713-23

Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland.

Serum protein glycosylation is known to be affected by pathological conditions, including cancer and inflammatory diseases. Pancreatic cancer patients would benefit from early diagnosis, as the disease is often detected in an advanced stage and has poor prognosis. Searching for changes in serum protein site-specific glycosylation could reveal novel glycoprotein biomarkers. We used Sambucus nigra lectin affinity chromatography to enrich α-2,6 sialylated tryptic N-glycopeptides from albumin-depleted sera of pancreatic cancer patients, acute pancreatitis patients, and healthy individuals, and compared their relative abundance using ultra performance LC-MS. Relative quantitation was done using the spectrum processing software MZmine. Identification was performed on the web-based tool GlycopeptideID, developed for in silico analysis of intact N-glycopeptides. Seventeen high-abundance serum proteins, mainly acute-phase proteins, and immunoglobulins, with total 27 N-glycosylation sites, and 62 glycoforms, were identified. Pancreatitis patient sera contained 38, and pancreatic cancer patients sera contained 13 glycoform changes with statistical significance (p < 0.05). In pancreatitis, up to tenfold changes were found in some glycoforms, and in pancreatic cancer, threefold. Analysis showed that the changes often concerned one or two, but not all, N-glycosylation sites in a specific glycoprotein. In conclusion, the analysis shows that pancreatic cancer, and acute pancreatitis are associated with changes in concentrations of intact sialylated N-glycopeptides derived from acute-phase proteins, and immunoglobulins, and that changes are site specific.
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http://dx.doi.org/10.1002/pmic.201300270DOI Listing
August 2014

Analytical and preanalytical validation of a new mass spectrometric serum 5-hydroxyindoleacetic acid assay as neuroendocrine tumor marker.

Clin Chim Acta 2014 Jan 8;428:38-43. Epub 2013 Nov 8.

HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.

Background: Serum 5-hydroxyindoleacetic acid (5-HIAA) could replace the determination of 24-h urinary 5-HIAA for diagnosis and follow-up of neuroendocrine tumors (NETs). We developed and validated a straightforward liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for serum 5-HIAA.

Methods: We used serum samples from healthy volunteers (n=136) and patients suspected or followed for NET (n=129). Samples were spiked with 5-HIAA-D2, extracted and quantified by LC-MS/MS. We studied the effects of sample storage, sample device, a meal and diurnal variation on serum 5-HIAA. Furthermore, we established a reference range for serum 5-HIAA and compared our assay with a urinary 5-HIAA HPLC assay and a commercial plasma chromogranin A (CgA) immunoassay.

Results: Our LC-MS/MS assay is sensitive (LOQ 5 nmol/L), has a wide assay range (5-10,000 nmol/L) and short analysis time (7 min). 5-HIAA in serum is stable for several days in various temperatures and during five freeze-thaw cycles. We found no diurnal variation (p ≥ 0.20) and a meal had no effect on serum 5-HIAA (p=0.89). We suggest an upper reference limit of 123 nmol/L for serum 5-HIAA. The area under curve (AUC) in receiver operator characteristics (ROC) analysis was 0.83 for urinary 5-HIAA, 0.81 for serum 5-HIAA and 0.76 for CgA, respectively.

Conclusions: The LC-MS/MS assay for serum 5-HIAA discriminates between healthy individuals and patients with NET and is well suited for the diagnosis and follow-up of NETs.
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http://dx.doi.org/10.1016/j.cca.2013.10.025DOI Listing
January 2014

Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction.

Proteome Sci 2013 Aug 1;11(1):38. Epub 2013 Aug 1.

Transplantation Laboratory, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.

Background: Cell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes. The surface sialylation pattern of cells is influenced by the cellular environment but also by the Golgi sialyltransferase activity and flux of metabolites through sialic acid producing pathways. Altered cell surface sialic acid patterns have been observed in several cancers and other pathological conditions. In this experiment we examined the cellular proteomic changes that occur in human embryonic kidney cells after 24 hours of sialic acid overproduction using N-Acetylmannosamine. We utilized high resolution mass spectrometry and label free protein quantification to characterize the relative changes in protein abundance as well as multiple reaction monitoring to quantify the cellular sialic acid levels.

Results: Using N-Acetylmannosamine we were able to induce sialic acid production to almost 70-fold compared to non-induced control cells. Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance. Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions. We also identified several physically interacting co-regulated proteins in the set of changed proteins.

Conclusions: In this experiment we show that increased metabolic flux through sialic acid producing pathway affects the abundance of several protein transport, epithelial adherens junction, signaling and metabolic pathway related proteins.
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http://dx.doi.org/10.1186/1477-5956-11-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750590PMC
August 2013

[Worms in prevention of allergy].

Duodecim 2013 ;129(10):1023-30

HYKS Iho- ja allergiasairaala ja Helsingin yliopisto, transplantaatiolaboratorio.

The increase in allergy and autoimmune diseases is suspected to be caused by the diminished contact of humans with microorganisms. Microorganisms, such as bacteria, viruses and their components, as well as parasitic worms modify the human defence system. The applicability of parasitic worms and their components to the prevention of allergy has been investigated. Most clinical studies have been carried out with whipworm eggs (Trichuris suis), because they do not cause a severe infection. There is preliminary evidence of their efficacy in inflammatory bowel diseases, but convincing evidence in the treatment of allergy is lacking.
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July 2013

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

PLoS One 2012 10;7(4):e34307. Epub 2012 Apr 10.

Department of Virology, Haartman Institute, University of Helsinki, Helsinki, Finland.

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034307PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323627PMC
October 2012

Relative quantification of several plasma proteins during liver transplantation surgery.

J Biomed Biotechnol 2011 10;2011:248613. Epub 2011 Dec 10.

HUSLAB, Helsinki University Central Hospital, 00290 Helsinki, Finland.

Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50-2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery.
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http://dx.doi.org/10.1155/2011/248613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237013PMC
June 2012

[Mechanisms and new innovations in hyposensitization].

Duodecim 2011 ;127(12):1289-95

Turun yliopisto, kliininen laitos ja TYKS:n allergiayksikkö, PL 52, 20521 Turku.

According to established view, the underlying factor in allergic immune response is imbalance between the number and function of allergen-specific CD4+ lymphocytes. In atopic allergy, type 2 helper T lymphocytes (Th2 cells) are elevated in relation to regulatory T lymphocytes (Treg cells). In healthy subjects the ratio is vice versa. It seems that tolerance generated through allergen-specific Treg cells is the immunologic reaction mode towards allergens in healthy non-atopic subjects. It has been found that hyposensitization will correct the imbalance of Th2 and Treg cells and restore the normal immune response to allergens.
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September 2011

[Are there enough medical scientists in Finland?].

Duodecim 2011 ;127(10):1003-9

Haartman-instituutti, Helsingin yliopisto ja HUSLAB.

Discussion within the scientific society and hospital communities has raised concerns of the current status and future of clinical research in Finland. One of the crucial future challenges is whether there are enough medical scientists that are able to perform clinical research and comprehend and manage medicine as a whole. In the article, the authors present suggestions for solving the problematic issues.
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July 2011

Allergy as an epithelial barrier disease.

Clin Transl Allergy 2011 Jun 10;1(1). Epub 2011 Jun 10.

Transplantation Laboratory & Infection Biology Research Program, Haartman Institute, University of Helsinki & Helsinki University Central Hospital, HUSLAB, Helsinki, Finland.

The objective of this review is to focus on putative modified epithelial functions related to allergy. The dysregulation of the epithelial barrier might result in the allergen uptake, which could be the primary defect in the pathogenesis of allergic reaction. We review the literature of the role of respiratory epithelium as an active barrier, how allergens are transported through it and how it senses the hostile environmental allergens and other dangerous stimuli.
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http://dx.doi.org/10.1186/2045-7022-1-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294629PMC
June 2011

Allergen interactions with epithelium.

Curr Opin Allergy Clin Immunol 2011 Feb;11(1):29-32

Transplantation Laboratory & Infection Biology Research Program, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.

Purpose Of Review: Allergies are a global health problem with rapidly increasing prevalence but still lacking pathogenetic knowledge or optimal treatment. The objective is to add to the conventional thinking that allergies are caused by overactive, mainly T-cell-mediated, immunological responses and thus to raise the putative role of altered epithelial functions.

Recent Findings: Birch pollen allergen was rapidly and actively transported through the respiratory epithelium via caveolar-dependent mechanisms only in patients allergic to birch pollen but not their healthy controls. Transcriptomic analyses showed that whereas healthy individuals raised a strong epithelial response after intranasal allergen challenge, the allergic patients had a reduced response. Thus allergies could also be due to hyporeactive responses on the epithelial level.

Summary: Epithelium has emerged as an active and complex organ with mechanical, biochemical and immunological functions. The increasing awareness that epithelium interacts actively with allergens might provide new targets for the prevention and management of allergy.
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http://dx.doi.org/10.1097/ACI.0b013e328342319eDOI Listing
February 2011

Allergens are transported through the respiratory epithelium.

Expert Rev Clin Immunol 2010 Jan;6(1):55-9

Transplantation Laboratory & Infection Biology Research Program, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.

We used a top-down approach with a wide repertoire of wet laboratory and in silico techniques for analyzing the pathogenesis of early events within the type I allergic reactions. We could show a caveolar-dependent transport of the birch pollen allergen through the respiratory epithelium of allergic patients but not of their healthy controls. The application of discovery-driven methodologies can provide new hypotheses worth further analyses of complex multifactorial diseases such as type I allergy.
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http://dx.doi.org/10.1586/eci.09.55DOI Listing
January 2010

Network analysis of single nucleotide polymorphisms in asthma.

J Asthma Allergy 2010 Dec 9;3:177-86. Epub 2010 Dec 9.

Transplantation Laboratory and Infection Biology Research Program, Haartman Institute, University of Helsinki, Helsinki;

Background: Asthma is a chronic inflammatory disease of the airways with a complex genetic background. In this study, we carried out a meta-analysis of single nucleotide polymorphisms (SNPs) thought to be associated with asthma.

Methods: The literature (PubMed) was searched for SNPs within genes relevant in asthma. The SNP-modified genes were converted to corresponding proteins, and their protein-protein interactions were searched from six different databases. This interaction network was analyzed using annotated vocabularies (ontologies), such as the Gene Ontology and Nature pathway interaction databases.

Results: In total, 127 genes with SNPs related to asthma were found in the literature. The corresponding proteins were then entered into a large protein-protein interaction network with the help of various databases. Ninety-six SNP-related proteins had more than one interacting protein each, and a network containing 309 proteins and 644 connections was generated. This network was significantly enriched with a gene ontology entitled "protein binding" and several of its daughter categories, including receptor binding and cytokine binding, when compared with the background human proteome. In the detailed analysis, the chemokine network, including eight proteins and 13 toll-like receptors, were shown to interact with each other. Of great interest are the nonsynonymous SNPs which code for an alternative amino acid sequence of proteins and, of the toll-like receptor network, TLR1, TLR4, TLR5, TLR6, TLR10, IL4R, and IL13 are among these.

Conclusions: Protein binding, toll-like receptors, and chemokines dominated in the asthma-related protein interaction network. Systems level analysis of allergy-related mutations can provide new insights into the pathogenetic mechanisms of disease.
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http://dx.doi.org/10.2147/JAA.S14459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047920PMC
December 2010

Mucosal eosinophils and l-selectin ligands are associated with invasive and noninvasive sinus surgery outcomes.

Am J Rhinol Allergy 2009 Jan-Feb;23(1):21-7

Department of Clinical Medicine University of Tampere, Tampere, Finland.

Background: Chronic rhinosinusitis (CRS) is characterized by persistent inflammation of the nasal and paranasal mucosa with numerous emigrated leukocytes. L-Selectin on leukocytes and its endothelial glycosylated ligands initiate leukocyte infiltration into inflamed tissues. Endoscopic sinus surgery (ESS) is the major approach for restoring sinus physiology after failure of conservative therapy; however, the effect of enlarging the maxillary sinus ostium is still unknown. Here, we compared two histological markers of local inflammation, the number of mucosal eosinophils, and the expression of endothelial L-selectin ligands, with clinical outcomes after enlarging or saving the maxillary sinus ostium.

Methods: Twenty-three patients with CRS underwent uncinectomy on one side and additional middle meatal antrostomy on the other side. Maxillary sinus mucosa biopsy specimens from these patients and nine healthy subjects were taken for immunohistochemical evaluations of the number of mucosal eosinophils and endothelial L-Selectin ligands. Also, symptoms and mucociliary clearance were measured.

Results: The postoperative reduction of the endothelial L-Selectin ligands was independent of the operation technique. There was a correlation between postoperative number of mucosal eosinophils and symptom score, which was also independent of the surgical technique. The postoperative decrease of mucosal eosinophils, as well as the correlation of the intraoperative eosinophils with the postoperative symptom score, was found only on antrostomy side.

Conclusion: ESS decreases the expression of endothelial L-Selectin ligands, which might lead to decreased eosinophil traffic into maxillary sinus mucosa, putatively more when enlarging the maxillary sinus ostium. Both intra- and postoperative low number of eosinophils seem to be indicators of good subjective recovery.
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http://dx.doi.org/10.2500/ajra.2009.23.3250DOI Listing
May 2009

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.

Blood 2009 Jul 9;114(3):733-41. Epub 2009 Apr 9.

Department of Biological and Environmental Sciences, Division of Biochemistry, University of Helsinki, Helsinki, Finland.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.
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http://dx.doi.org/10.1182/blood-2009-03-210237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713463PMC
July 2009

Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients.

J Allergy Clin Immunol 2009 Jul 2;124(1):135-142.e1-21. Epub 2009 Apr 2.

Transplantation Laboratory and Infection Biology Research Program, Haartman Institute, University of Helsinki, Helsinki FI-00014, Finland.

Background: Previous work in type I pollen allergies has focused on aberrant immunoresponses.

Objective: Our systems-level analyses explore the role of epithelium in early pathogenesis of type I allergic reactions.

Methods: We began top-down analyses of differences in human nasal epithelial cells and biopsy specimens obtained from patients with birch allergy and healthy control subjects in the resting state and after intranasal in vivo birch pollen challenges. Immunohistochemistry, immunotransmission electron microscopy, mass spectrometry, transcriptomics, and integration of data to a pathway were conducted.

Results: Bet v 1 allergen bound to epithelium immediately after in vivo birch pollen challenge during winter only in allergic individuals. It also travelled through epithelium with caveolae to mast cells. Sixteen unique proteins were found to bind to the Bet v 1 column only in lysates from allergic epithelial cells; 6 of these were caveolar and 6 were cytoskeletal proteins. The nasal epithelial transcriptome analysis from allergic and healthy subjects differed during the winter season, and these subjects also responded differentially to birch pollen challenge. Within this pollen-induced response, the gene ontology categories of cytoskeleton and actin cytoskeleton were decreased in allergic patients, whereas the actin-binding category was enriched in healthy subjects. Integration of microscopic, mass spectrometric, and transcriptomic data to a common protein-protein binding network showed how these were connected to each other.

Conclusion: We propose a hypothesis of caveolae-dependent uptake and transport of birch pollen allergen in the epithelium of allergic patients only. Application of discovery-driven methodologies can provide new hypotheses worth further analysis of complex multifactorial diseases, such as type I allergy.
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http://dx.doi.org/10.1016/j.jaci.2008.11.048DOI Listing
July 2009

De novo glycan structure search with the CID MS/MS spectra of native N-glycopeptides.

Glycobiology 2009 Jul 6;19(7):707-14. Epub 2009 Mar 6.

Transplantation Laboratory & Infection Biology Research Program, Haartman Institute, University of Helsinki, Finland.

The aim of our study is to automatically analyze the glycan and peptide structures of N-glycopeptides without a need to release glycans from the glycopeptides. Our wet laboratory raw data represent a series of MS/MS mass spectra obtained from a reverse-phase liquid chromatography run of size-exclusion-enriched tryptic-digested glycopeptides from glycoproteins. The MS/MS spectra are first analyzed in order to identify glycosylated peptides and N-glycan monosaccharide compositions present on each glycopeptide. We further developed a Branch-and-Bound algorithm to search de novo N-glycan structures, i.e., monosaccharide compositions and their ordered sequences from native glycopeptides. Our de novo algorithm is based on iterative growth and selection of a population of glycan structures and it does not use databases of known glycan structures. We validate the algorithm with (i) in silico-generated spectra, with or without deteriorating deletions, (ii) with a purified glycoprotein transferrin, and (iii) with a complex mixture of N-glycopeptides enriched from human plasma. Our Branch-and-Bound algorithm depicted glycan structures from all the above-mentioned three input data types. Due to the large diversity of glycan structures, the results typically contained several proposed structures matching almost equally well to the spectra. In conclusion, this algorithm automatically identifies glycopeptides and their structures from the MS/MS spectra and thus greatly reduces the number of possible glycan structures from the vast amount of potential ones.
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http://dx.doi.org/10.1093/glycob/cwp034DOI Listing
July 2009

Concordant gene regulation related to perturbations of three GDP-mannose-related genes.

FEMS Yeast Res 2009 Feb;9(1):63-72

Medicel Ltd., Helsinki, Finland.

Glycosylation of proteins is one of the most crucial post-translational modifications. In order to access system-level and state-dependent data related to the regulation of glycosylation events, we cultivated yeast cell strains each harboring a selected conditional knockdown construct for a gene (either SEC53, VRG4 or DPM1) related to GDP-mannose synthesis or its utilization in glycan biosynthesis. In order to carry this out efficiently, we developed automated sampling from bioreactor cultivations, a collection of in silico workflows for data analysis as well as their integration into a large data warehouse. Using the above-mentioned approaches, we could show that conditional knocking down of transcripts related to GDP-mannose synthesis or transportation led to altered levels of over 300 transcripts. These transcripts and their corresponding proteins were characterized by their gene ontology (GO) annotations, and their putative transcriptional regulation was analyzed. Furthermore, novel pathways were generated indicating interactions between GO categories with common proteins, putative transcriptional regulators of such induced GO categories, and the large protein-protein interaction network among the proteins whose transcripts indicated altered expression levels. When these results are always added to an ever-expanding data warehouse as annotations, they will incrementally increase the knowledge of biological systems.
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http://dx.doi.org/10.1111/j.1567-1364.2008.00461.xDOI Listing
February 2009

Differential metabolic consequences of fumarate hydratase and respiratory chain defects.

Biochim Biophys Acta 2008 May 14;1782(5):287-94. Epub 2008 Feb 14.

Program of Molecular Neurology Biomedicum-Helsinki, Finland.

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1alpha was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.
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http://dx.doi.org/10.1016/j.bbadis.2008.01.008DOI Listing
May 2008

N-glycoproteomics - an automated workflow approach.

Glycobiology 2008 Apr 13;18(4):339-49. Epub 2008 Feb 13.

MediCel, Haartmaninkatu 8, FIN-00290 Helsinki, Finland.

Glycan decorations dictate protein functions and thus have crucial importance in life sciences. Previously glycoprotein analysis was mainly focused on the analysis of the liberated glycans allowing detailed structural, but lacking positional information. Analysis of intact glycopeptides required purified glycoproteins and manual interpretation of spectra. We developed an approach where mixtures of native glycopeptides were analyzed with tandem mass spectrometry and the spectra were analyzed with automated in silico workflows. The latter included combination of the original spectra, generation of a human N-glycopeptide library, matching the glycopeptide spectra to the theoretical peptide fragments, scoring the observations, predicting the glycan composition, which were then matched against the observed spectra, statistical validation of the results with target-decoy filtering, and finally the calculation of glycan structures. We verified this approach with the 150 serotransferrin glycopeptide spectra, where we automatically generated 10(5) putative interpretations from >10(9) theoretical glycopeptides. After scoring 62 glycopeptide spectra obtained validated interpretation with concomitant amino acid sequences, glycan compositions, and structures. When applying this method to an unknown mixture of human plasma glycoproteins we identified 80 glycopeptides with their glycan compositions or structures. Instead of weeks and months of interpretation work of mass spectrometry files our automated workflow can be executed in few hours and provide information concomitantly from both the amino acid and glycan moieties of intact glycopeptides in mixtures. No advanced computational skills were needed to use these preformed and tested workflows. In case users want to add complexity to the analysis they are allowed to alter all parameters and rebuild the workflows.
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http://dx.doi.org/10.1093/glycob/cwn013DOI Listing
April 2008

Differential gene expression of GDP-L-fucose-synthesizing enzymes, GDP-fucose transporter and fucosyltransferase VII.

APMIS 2006 Jul-Aug;114(7-8):539-48

Rational Drug Design program, Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, Helsinki, Finland.

L-fucose is a fundamental monosaccharide component of many mammalian glycoproteins and glycolipids. Fucosylation requires GDP-L-fucose as a donor of fucose and a specific fucosyltransferase (Fuc-T) to catalyze the transfer of L-fucose to various lactosamine acceptor molecules. The biosynthesis of GDP-L-fucose consists of two pathways. The constitutively active de novo pathway involves conversion of cellular GDP-D-mannose to GDP-L-fucose by GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (FX). In the alternative biosynthetic pathway, in the salvage metabolism, L-fucokinase (Fuk) synthesizes L-fucose-1-phosphate from free fucose. L-fucose-1-phosphate is further catalyzed to GDP-L-fucose by GDP-L-fucose pyrophosphorylase (Fpgt). GDP-L-fucose, synthesized in the cytosol, is translocated to the Golgi for fucosylation by a specific GDP-fucose transporter (FUCT1). Glycans that contain alpha(1,3)-fucosylated modifications, e.g. sialyl Lewis X-type glycans, have an important role in inflammation and in tumorigenesis. We studied the mRNA expression levels of GDP-L-fucose-synthesizing enzymes, GDP-fucose transporter and fucosyltransferase VII by quantitative real-time PCR in mouse endothelial cells, macrophages and lymphoid tumor cells. Moreover, the expression of the same transcripts was detected in acute inflammation using rat kidney allograft as model system. Our results indicate the simultaneous upregulation of the GDP-L-fucose synthesizing enzymes of the de novo pathway, GDP-fucose transporter and fucosyltransferase VII in inflammation and in tumorigenesis.
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http://dx.doi.org/10.1111/j.1600-0463.2006.apm_461.xDOI Listing
October 2006

Cloning and expression of rat fucosyltransferase VII at sites of inflammation.

APMIS 2005 Sep;113(9):613-20

Rational Drug Design program, Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, Helsinki, Finland.

The sialyl Lewis x (NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium. The final step in the sialyl Lewis x synthesis is catalyzed by alpha1-3-fucosyltransferase, which transfers fucose to sialylated type 2 chain. We report the cloning of rat alpha1-3-fucosyltransferase gene (rFUT) isolated from rat lymph node and kidney allograft. The rFUT is expressed as two splice variants, but only the long one showed enzymatic activity towards sialylated lactosamine. Also flow cytometry analysis with the sLex mAbs indicated that the cloned rFuc-T was a functional enzyme and a member of the Fuc-TVII family. The rFuc-TVII mRNA expression level was strongly enhanced during acute inflammatory reaction induced by kidney allograft rejection, which could be detected by in situ hybridization and quantitative real-time PCR.
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http://dx.doi.org/10.1111/j.1600-0463.2005.apm_279.xDOI Listing
September 2005

Endothelial L-selectin ligands in sinus mucosa during chronic maxillary rhinosinusitis.

Am J Respir Crit Care Med 2005 Jun 11;171(12):1350-7. Epub 2005 Mar 11.

Department of Eye, Ear and Oral Diseases, Tampere University Hospital and University of Tampere, Tampere, Helsinki.

Rationale: Chronic rhinosinusitis is characterized by persistent inflammation of the nasal and paranasal mucosa with numerous emigrated leukocytes. L-selectin on leukocytes and its endothelial glycosylated ligands initiate organ-specific leukocyte infiltration into inflamed tissues.

Objectives: The purpose of this study was to evaluate the endothelial expression of functionally active endothelial L-selectin ligands, sulfated sialyl Lewis x, in maxillary sinus mucosa from patients with chronic rhinosinusitis and from normal control subjects.

Methods: Maxillary sinus mucosa specimens (116) were obtained surgically and immunohistochemically stained with monoclonal antibodies detecting sialyl Lewis x or sulfated extended core 1 lactosamines. The severity of the inflammation was determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of the specimens.

Measurements And Main Results: The percentage of vessels expressing endothelial sulfated sialyl Lewis x epitopes increased during chronic rhinosinusitis compared with uninflamed control tissue, especially in patients with additional allergic rhinitis, and decreased in specimens from aspirin-intolerant patients with preoperative oral corticosteroid treatment. In addition, the expression level of endothelial sulfated sialyl Lewis x epitopes and the number of mucosal eosinophils correlated with the severity of the inflammation, and decreased in specimens taken 9 months postoperatively compared with intraoperative samples, especially in patients with intranasal corticosteroid treatment.

Conclusions: Our results suggest that functionally active L-selectin ligands might guide leukocyte traffic into maxillary sinus mucosa preferentially in patients with severe findings of chronic maxillary rhinosinusitis, thus leading to aggravation of the inflammation.
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http://dx.doi.org/10.1164/rccm.200406-775OCDOI Listing
June 2005

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex.

FEMS Yeast Res 2005 Feb;5(4-5):341-50

Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland.

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.
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http://dx.doi.org/10.1016/j.femsyr.2004.11.007DOI Listing
February 2005

Excess mannose limits the growth of phosphomannose isomerase PMI40 deletion strain of Saccharomyces cerevisiae.

J Biol Chem 2004 Dec 1;279(53):55737-43. Epub 2004 Nov 1.

MediCel Ltd., Haartmaninkatu 8, FIN-00290 Helsinki, Finland.

Phosphomannose isomerase (PMI40) catalyzes the conversion between fructose 6-phosphate and mannose 6-phosphate and thus connects glycolysis, i.e. energy production and GDP-mannose biosynthesis or cell wall synthesis in Saccharomyces cerevisiae. After PMI40 deletion (pmi(-)) the cells were viable only if fed with extracellular mannose and glucose. In an attempt to force the GDP-mannose synthesis in the pmi(-) strain by increasing the extracellular mannose concentrations, the cells showed significantly reduced growth rates without any alterations in the intracellular GDP-mannose levels. To reveal the mechanisms resulting in reduced growth rates, we measured genome-wide gene expression levels, several metabolite concentrations, and selected in vitro enzyme activities in central metabolic pathways. The increasing of the initial mannose concentration led to an increase in the mannose 6-phosphate concentration, which inhibited the activity of the second enzyme in glycolysis, i.e. phosphoglucose isomerase converting glucose 6-phosphate to fructose 6-phosphate. As a result of this limitation, the flux through glycolysis was decreased as was the median expression of the genes involved in glycolysis. The expression levels of RAP1, a transcription factor involved in the regulation of the mRNA levels of several enzymes in glycolysis, as well as those of cell cycle regulators CDC28 and CLN3, decreased concomitantly with the growth rates and expression of many genes encoding for enzymes in glycolysis.
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http://dx.doi.org/10.1074/jbc.M410619200DOI Listing
December 2004

Inflammation-induced transcriptional regulation of Golgi transporters required for the synthesis of sulfo sLex glycan epitopes.

Glycobiology 2004 Dec 21;14(12):1285-94. Epub 2004 Jul 21.

Rational Drug Design Program, Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, P.O. Box 63, FIN-00014 University of Helsinki, Finland.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.
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http://dx.doi.org/10.1093/glycob/cwh131DOI Listing
December 2004

Monitoring leukocyte traffic in vivo into human delayed-type hypersensitivity reaction.

J Immunol Methods 2004 May;288(1-2):81-9

Rational Drug Design Program, Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, P.O. Box 63, Helsinki FIN-00014, Finland.

Leukocyte traffic from blood to sites of inflammation has been elaborately studied in animal models and in vitro. However, to date, little understanding has accumulated on the process in humans in vivo. A noninvasive light confocal microscopy technique has enabled us to image leukocyte rolling, arrest and transmigrated cells in vivo in human tissues. In the current study, a delayed-type hypersensitivity (DTH) reaction was elicited in the lower lip of healthy, Bacille Calmette-Guerin (BCG)-vaccinated volunteers by injection of respective purified protein derivate (PPD), mimicking the classic cutaneous Mantoux reaction. Subjects were imaged with real-time confocal microscopy at baseline and 2, 4, 24 and 48 h after the injection. The number of rolling leukocytes did not increase significantly until at 48 h. Even then, rolling cells were seen in only a minority (23%) of blood vessels. The frequency of engaged blood vessels was, nevertheless, significantly greater than at baseline. As the inflammation generated with this challenge was mild, transmigrated leukocytes could be detected in the confocal microscopy only occasionally. Histology of biopsies taken immediately after imaging at 48 h showed a T cell-dominated leukocyte population at the site of the DTH reaction. We have thus developed a method to monitor noninvasively leukocyte traffic in vivo in human subjects during the classic inflammatory model of delayed-type hypersensitivity reaction.
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http://dx.doi.org/10.1016/j.jim.2004.02.009DOI Listing
May 2004

Direct in vivo monitoring of acute allergic reactions in human conjunctiva.

J Immunol 2004 Mar;172(5):3235-42

Department of Ophthalmology, Helsinki University Central Hospital Laboratory Diagnostics, Helsinki, Finland.

Immediate allergic reactions are initiated by allergen-induced, specific IgE-mediated mast cell degranulation and involve leukocyte recruitment into the inflamed site. We compared conjunctival signs, symptoms, and in vivo leukocyte rolling and extravasation into sites of inflammation in five patients allergic to birch pollen and in 10 nonallergic controls who received a challenge to birch allergen or histamine. Both the specific allergen in allergic patients and histamine, both in patients and in healthy controls, induced symptoms and signs of an immediate allergic reaction together with leukocyte rolling within the conjunctival blood vessels. However, only allergen, not histamine, caused leukocyte extravasation into the site of inflammation in the allergic patients. Allergen also increased expression of endothelial P-selectin in conjunctival vessels and slowed the rolling of leukocytes which is required for their extravasation from blood circulation into the target tissue. Finally, i.v. heparin strongly reduced the number of slowly rolling cells during allergen- or histamine-induced reactions and this can probably hinder the leukocyte extravasation after allergen exposure. These findings suggest that slow rolling is required for leukocyte extravasation in acute allergic reactions, and it can be inhibited by heparin in vivo in therapeutically relevant conditions.
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http://dx.doi.org/10.4049/jimmunol.172.5.3235DOI Listing
March 2004

Biosynthesis of 6-deoxyhexose glycans in bacteria.

Glycobiology 2004 Mar 23;14(3):1R-15R. Epub 2003 Dec 23.

Rational Drug Design Program and Department of Bacteriology and Immunology, Biomedicum and Haartman Institute, PO Box 63, FIN-00014 University of Helsinki, Helsinki, Finland.

After the breakthroughs in genomic sequencing, one of the next challenges remains to understand the molecular biology of other classes of biomolecules, such as protein and lipids, many of which carry specific glycomodification when mediating their biological functions. This review focuses on the 6-deoxyhexose biosynthesis of cell surface glycans of three Gram-negative pathogens, Helicobacter pylori, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans serotype a. 6-Deoxysugars are important functional components of cell surface glycans, and their biosynthetic pathways might be suitable targets for novel interventions of antibacterial chemotherapy.
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http://dx.doi.org/10.1093/glycob/cwh040DOI Listing
March 2004