N-Glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N‐glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and ∼4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2‐inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylate

Tumor Lymphangiogenesis and Metastasis to Lymph Nodes Induced by Cancer Cell Expression of Podoplanin

The membrane glycoprotein podoplanin is expressed by several types of human cancers and might be associated with their malignant progression. Its exact biological function and molecular targets are unclear, however. Here, we assessed the relevance of tumor cell expression of podoplanin in cancer metastasis to lymph nodes, using a human MCF7 breast carcinoma xenograft model. We found that podoplanin expression promoted tumor cell motility in vitro and, unexpectedly, increased tumor lymphangiogenesis and metastasis to regional lymph nodes in vivo, without promoting primary tumor growth. Importantly, high cancer cell expression levels of podoplanin correlated with lymph node metastasis and reduced survival times in a large cohort of 252 oral squamous cell carcinoma patients. Based on comparative transcriptional profiling of tumor xenografts, we identified endothelin-1, villin-1, and tenascin-C as potential mediators of podoplanin-induced tumor lymphangiogenesis and metastasis. These unexpected findings identify a novel mechanism of tumor lymphangiogenesis and metastasis induced by cancer cell expression of podoplanin, suggesting that reagents designed to interfere with podoplanin function might be developed as therapeutics for patients with advanced cancer

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and similar to 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated

Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin

Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc—although produced specifically in the skin—entered the blood circulation and induced disseminated intravascular coagulation, characterized by microthrombi in most organs and by thrombocytopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, keratin 14 podoplanin-Fc mice represent a novel genetic animal model of disseminated intravascular coagulation