Karl Tryggvason

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator☆

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.

Primary Structure, Developmental Expression, and Immunolocalization of the Murine Laminin α4 Chain

The complete primary structure of the mouse laminin α4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature α4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin α4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin α4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.

Characterization of Recombinant Soluble Macrophage Scavenger Receptor MARCO

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.

Molecular cloning and tissue-specific expression of a novel murine laminin gamma3 chain.

A novel laminin γ3 chain was identified from the expressed sequence tag data base at the National Center for Biotechnology Information. A complete cDNAderived peptide sequence reveals a 1592-amino acid-long primary translation product, including a tentative 33-amino acid-long signal peptide. Comparison with the laminin γ1 chain predicts that the two polypeptides have equal spatial dimensions. In addition, the well conserved domains VI and III(LE4) predict that γ3 containing laminins are able to integrate to the laminin network and also via nidogen connect to other protein networks in the basement membranes. Combination of Northern analysis and in situ hybridization experiments indicate that expression of the γ3 chain is highly tissue- and cell-specific, being significantly strong in capillaries and arterioles of kidney as well as in interstitial Leydig cells of testis.

Diseases of the Glomerular Filtration Barrier: Alport Syndrome and Congenital Nephrosis (NPHS1)

Publisher Summary This chapter focuses on Alport syndrome (AS) and congenital nephrosis of the Finnish type (NPHS1), the two best characterized genetic diseases of the glomerular capillary wall. AS is caused by mutations in genes coding for type IV collagen of the glomerular basement membrane (GBM). Cases of AS can be classified into X-linked and autosomal forms. Mutations in AS result in abnormal collagen chain composition and irregular ultrastucture of the GBM. The typical clinical features of AS are progressive nephritis, sensorineural hearing loss, and ocular changes. The diagnostic criteria used for differential diagnosis include a positive family history of hematuria with or without progression to end stage renal disease (ESRD); progressive sensorineural hearing loss; characteristic ocular changes (lenticonus and/or maculopathy); typical ultrastructural changes in the GBM; diffuse and abnormal GBM distribution of the α3, α4, and α5 chains of type IV collagen; and esophageal leiomyomatosis. NPHS1 is a rare disease caused by mutations in NPHS1, encoding a cell adhesion protein nephrin. Nephrin is synthesized by podocytes and is localized at the slit diaphragm area of the glomerular capillary wall. Infants with NPHS1 do not have any major nonrenal malformations; however, minor functional disorders in the central nervous system and cardiac hypertrophy are common during the nephrotic stage. Minor mutations in NPHS1 lead to atypical and milder forms of nephrotic syndrome. Children with classical NPHS1 are treated successfully with active protein and nutritional support, followed by bilateral nephrectomy, dialysis, and renal transplantation.