Mouse vascular adhesion protein 1 is a sialoglycoprotein with enzymatic activity and is induced in diabetic insulitis

Abstract

The continuous recirculation of lymphocytes requires an adequate expression and function of the molecules mediating the cellular interactions between endothelium and lymphocytes. Human vascular adhesion protein 1 (hVAP-1) is an endothelial cell adhesion molecule that mediates the binding of lymphocytes to venules in peripheral lymph nodes as well as at sites of inflammation. Recently the mouse homologue of hVAP-1 has been cloned. It is a previously unknown molecule with a significant sequence identity to copper-containing amine oxidases. Besides the sequence, very little is known about the expression, structure, and function of mouse VAP-1 (mVAP-1). In this study we demonstrate that mVAP-1 is prominently expressed in endothelial and smooth muscle (but not in other types of muscle cells), as well as in adipocytes. mVAP-1 is a 220-kd homodimeric sialoglycoprotein that displays cell-type-specific differences in glycosylation. The expression of mVAP-1 is induced on inflammation in the vessels of the endocrine pancreas during the development of insulitis, and the up-regulation correlates with the extent of the lymphocytic infiltrate. In general, different mouse strains displayed very similar VAP-1 expression, but the small differences seen in liver and gut suggest that immunostimulation may modulate VAP-1 synthesis in extrapancreatic organs as well. Finally, we show that mVAP-1 has a monoamine oxidase activity against naturally occurring substrates, implying a role in the development of vasculopathies. These data show that mVAP-1 and hVAP-1 are very similar molecules that nevertheless have certain marked differences in expression, biochemical structure, and substrate specificity. Thus mVAP-1 is a novel inflammation-inducible mouse molecule that has a dual adhesive and enzymatic function.

Figure 1.

mAb TK10-79 recognizes recombinant mVAP-1. mVAP-1 cDNA or mock transfected CHO cells were stained with mAbs Hermes-1 (negative control) or with TK 10-79 (against mVAP-1) and analyzed by FACS. The x axis is the fluorescence intensity on a log-scale, and the y axis is the relative number of cells. A positive staining with mAb TK10-79 is obtained only after fixation of the transfectants with methanol.

Figure 2.

Tissue distribution of mVAP-1. Staining of formalin-fixed paraffin-embedded mouse tissues with a negative control mAb Hermes-1 are shown in the last (narrower) micrograph of each series, and anti-mVAP-1 MAb TK10-79 stainings are shown in the first or first two micrographs of each series. A: Gut. Bw, bowel wall; Mm, muscularis mucosae. Arrows indicate lamina proporia vessels. B: Peyer’s patch. Arrowheads point to two mVAP-1-positive HEVs. C: Spleen. wp, white pulp. Arrows indicate two mVAP-1-positive splenic vessels. D: Bone. Sinusoidal vessels of bone marrow in femur are pointed out by arrows. Tr, bone trabeculae. E: Thymus. Arrows point to mVAP-1-positive vessels at the corticomedullary junction. F: Lung. Bronchus epithelium (curved arrow) lacks mVAP-1, whereas the smooth muscle layer of terminal bronchii (arrowheads) and endothelium of large veins (arrows) are mVAP-1 positive. G: Heart. Capillary endothelium (arrows) and larger vessels are mVAP-1 positive. H: Kidney. mVAP-1 is absent from glomeruli (gl), tubuli (one outlined with a dashed line), and intertubular vessels (arrows) but present on afferent arterioles (arrowheads). I: Liver. Sinusoidal endothelial cells are very faintly mVAP-1 positive (arrowheads), whereas central veins (arrow) are strongly mVAP-1 positive. J: White fat. The original magnifications were ×100 (A1, C2, E2, H1), ×200 (B1 and 2, C1, F1 and 3, G1), and ×400 in the others.

Figure 2.

Tissue distribution of mVAP-1. Staining of formalin-fixed paraffin-embedded mouse tissues with a negative control mAb Hermes-1 are shown in the last (narrower) micrograph of each series, and anti-mVAP-1 MAb TK10-79 stainings are shown in the first or first two micrographs of each series. A: Gut. Bw, bowel wall; Mm, muscularis mucosae. Arrows indicate lamina proporia vessels. B: Peyer’s patch. Arrowheads point to two mVAP-1-positive HEVs. C: Spleen. wp, white pulp. Arrows indicate two mVAP-1-positive splenic vessels. D: Bone. Sinusoidal vessels of bone marrow in femur are pointed out by arrows. Tr, bone trabeculae. E: Thymus. Arrows point to mVAP-1-positive vessels at the corticomedullary junction. F: Lung. Bronchus epithelium (curved arrow) lacks mVAP-1, whereas the smooth muscle layer of terminal bronchii (arrowheads) and endothelium of large veins (arrows) are mVAP-1 positive. G: Heart. Capillary endothelium (arrows) and larger vessels are mVAP-1 positive. H: Kidney. mVAP-1 is absent from glomeruli (gl), tubuli (one outlined with a dashed line), and intertubular vessels (arrows) but present on afferent arterioles (arrowheads). I: Liver. Sinusoidal endothelial cells are very faintly mVAP-1 positive (arrowheads), whereas central veins (arrow) are strongly mVAP-1 positive. J: White fat. The original magnifications were ×100 (A1, C2, E2, H1), ×200 (B1 and 2, C1, F1 and 3, G1), and ×400 in the others.

Figure 3.

The expression of mVAP-1 and MAdCAM-1 in pancreas. A, B: MAdCAM-1 is expressed both on vessels within the inflamed islets (IS) and on periislet vessels of pancreas from a 12-week NOD mouse. C, D: mVAP-1 staining of the same islets as in A and B in serial sections. Note that the vessels marked by arrows coexpress MAdCAM-1 and VAP-1. Original magnifications: (A, C) ×200; (B, D) ×400.

Figure 4.

Lymphocyte infiltration correlates with the expression of mVAP-1 in islet vessels. A: In islet (IS) with no/very low lymphocyte infiltration the expression of mVAP-1 is undetectable, whereas islets (B) with a high number of lymphocytes have strongly mVAP-1-positive vessels (arrow). Original magnification, ×400.

Figure 5.

TK10-79 recognizes a 110/220 kd molecule in different mouse tissues. Lysates from different mouse organs were separated by SDS-PAGE and thereafter subjected to immunoblotting with the indicated mAbs. A: Under nonreducing conditions a 220-kd band (arrow) can be detected, whereas the monomeric 110-kd band is detectable only from adipose tissue and smooth muscle lysate (in overexposures the 110-kd band can be seen also from lanes 1 and 3–13). hVAP-1 in smooth muscle lysate (arrowhead) is smaller than the corresponding mouse antigen (lane 9). B: Under reducing conditions, the monomeric ∼110-kd form of mVAP-1 (arrowhead) and a high-molecular-weight smear from mouse smooth muscle lysate are detected. Molecular weight standards are listed on the left.

Figure 6.

mVAP-1 is a glycoprotein. Lysates from the indicated mouse tissues were subjected to different glycosidase digestions before SDS-PAGE and immunoblotting with mAb TK10-79 under reducing conditions. All of the negative control stainings with Hermes-1 were negative. −, nontreated; sial, V. cholerae sialidase; O-Glyc, endo-α-N-acetylgalactosaminidase; N-Glyc, peptide:N-glycanase F is the name of the enzyme. The molecular weight standards are listed on the left.

Figure 7.

Substrate specificity of mVAP-1. Formation of labeled aldehyde product from radioactive benzylamine (Bz) in a mVAP-1-catalyzed reaction in the absence (−) or in the presence of competing amines was measured using scintillography. Quantification of the results (mean activity in cpm ± SEM) was obtained from two independent enzyme assays, each performed in duplicate with two different mVAP-1 CHO lines. PEA, phenylethylamine.