Risto Renkonen

A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.

Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.

Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

Endothelial and epithelial expression of sialyl Lewisx and sialyl Lewisa in lesions of breast carcinoma

Tumor cells can invade and generate metastasis via either lymphatics or blood vessels. When tumor cells are circulating in the blood, they must adhere to the vessel wall, which is lined by endothelium, before they can extravasate and form new metastases. Several families of adhesion molecules have been identified to play a role in the extravasation cascade. Selectins and their sialyl Lewisx and/or sialyl Lewisa (sLex and sLea, respectively) containing ligands play an initiating role in this cascade; we have now analyzed their role in the generation of metastatic breast carcinoma lesions. We examined expression of endothelial E‐ and P‐selectin, expression of epithelial and endothelial sLex and sLea normal tissues compared with primary and metastatic breast in carcinoma lesions within individual patients. While normal breast epithelial cells do not express sLex or sLea, epithelial expression of these oligosaccharide epitopes was enhanced in primary breast carcinoma lesions. Furthermore, epithelial expression levels of sLex and/or sLea were even higher in most patients (9 of 12) who had metastatic compared with primary lesions. We show that endothelia in primary lesions express more sLex than in normal tissue and that metastatic lesions express even higher amounts of sLex compared with primary lesions. The expression of P‐ and E‐selectin was also greatly enhanced in tumor‐bearing tissue compared with normal tissue. Our data support the hypothesis that while they are circulating in the blood, sLex‐ and/or sLea‐expressing carcinoma cells have a higher probability for extravasation at sites where the endothelium expresses E‐ and P‐selectin and for generation of new metastases. Int. J. Cancer 74:296‐300, 1997.

Recombinant E-selectin-protein mediates tumor cell adhesion via sialyl-Lea and sialyl-Lex

E-selectin (previously known as ELAM-1) is one of the adhesion molecules expressed on activated endothelium. Here we show that HL-60 cells express sialyl-Le(x), but not Sialyl-Le(a) on their surface, a colon carcinoma cell line COLO 205 express both these epitopes and another colon carcinoma COLO 320 does not express either one of them. HL-60 and COLO 205 cell adhere strongly to E-selectin coated microwells, whereas COLO 320 does not adhere at all to E-selectin. Finally we provide evidence that monoclonal anti-sialyl-Le(x) can abolish part of the adherence of HL-60 cells to recombinant E-selectin. The adherence of COLO 205 cells can be decreased by either monoclonal anti-sialyl-Le(a) or anti-sialyl-Le(x) antibodies. These results indicate that cell-associated sialylated carbohydrate moieties can act as ligands for recombinant E-selectin.

The inflammatory mechanisms of allograft rejection

If one wishes to describe the rejection process in biologicai terms, it may be divided into the following, largely overlapping components: (a) induction of rejection, i.e., how does the alien graft induce the antiallograft immune response, (b) the immune response towards the graft, and (c) the inflammatory response of rejection and regulation of the inflammation by the immune response. Finally one should consider (d) the effector mechanisms in situ, i.e., how do the inflammatory cells, together with the antibody, destroy the graft.

Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites.

Inflammatory diseases are characterized by the leukocyte infiltration into tissues. L-selectin on lymphocytes and its endothelial glycosylated ligands are instrumental in the initiation of lymphocyte extravasation. Immunohistochemical stainings with monoclonal antibodies against functionally active glycan-decorated L-selectin ligands, ie, sialyl-Lewis x (sLex, 2F3, and HECA-452) or sulfated extended core 1 lactosamine (MECA-79), were performed on more than 400 specimen representatives for thyroiditis, myocarditis, psoriasis, vasculitis, ulcerative colitis, and their corresponding noninflamed tissues. The endothelial expression of sLex or sulfo sLex glycans in postcapillary venules was either absent or low in control tissues. The de novo induction of endothelial expression of sLex or sulfo sLex glycans was detected in all inflamed tissues. Furthermore, each organ carried its own modification of sLex or sulfo sLex glycans, ie, zip code. Our results suggest that these zip code glycans may provide means for organ selective leukocyte traffic that could be used in selective leukocyte traffic inhibition.

TNFa‐Induced Expression of Endothelial Adhesion Molecules, ICAM‐1 and VCAM‐1, is Linked to Protein Kinase C Activation

The role of protein kinase C (PKC) in TNFα‐induced activation of endothelial adhesion molecules ICAM‐1 and VCAM‐1 was analysed. Phorbol myristate acetate, which is known to activate PKC, was able lo mimic TNFα‐induced up‐regulation of ICAM‐1 and partly also VCAM‐1 expression. Similarly a PKC inhibitor, H7, but not another kinase inhibitor. HA1004, inhibited TNFα‐induced enhancement of ICAM‐1 expression at both the mRNA and the protein level. Moreover we were able to measure a transient PKC activation peak at 16 min after TNFα induction in endothelial cells analysed by phorbol‐dibutyrate binding. These results indicate that the TNFα‐induced effect on the regulation of endothelial adhesion molecule expression is at least partly mediated by PKC activation.

N-Glycoproteomics – An automated workflow approach

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.

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin.