Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.

EMILIN (elastin microfibril interface located protein)is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-dayold embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMI-LIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil ␣ helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of ␤-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

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The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded ␤-sandwich fold of the gC1q domain is reduced to nine ␤ strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the ␣4␤1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of ␣4␤1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu 933 ). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-␣4␤1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.EMILIN1 (elastin microfibril interface located protein 1) is a secreted extracellular matrix multidomain glycoprotein (1, 2). It is characterized by a unique arrangement of structural domains, including the elastin microfibril interface domain at the N terminus, an ␣-helical domain predicted to form a coiledcoil structure in the central part of the molecule, a short collagenous sequence, and a region homologous to the globular domain of C1q (gC1q domain) 4 at the C-terminal end (3, 4). Although the role of the coiled-coil region has not yet been elucidated, it has conclusively been demonstrated that EMILIN1 interacts with pro-tumor growth factor-␤ (5) through the elastin microfibril interface domain (6). EMILIN1 deficiency causes systemic arterial hypertension, and the expression of EMILIN1 at physiological levels by binding to pro-tumor growth factor-␤ prevents its maturation by protein convertases (5). Thus, EMILIN1 favorably located at the subendothelium of blood vessels is a new specific antagonist of tumor growth factor-␤, and the function of this constituent of elastic tissues is linked to the pathogenesis of hypertension. The C-terminal gC1q domain is involved in the oligomerization of EMILIN1 (7), in cell adhesion and migration via interaction with the ␣4␤1 integrin (8), and in trophoblast invasion (9).The gC1q signature is found in a variety of proteins, and the essential features of the specific structure-function relationship were recognized with the elucidation of the crystal structure of the homotrimeric gC1q domain of mouse ACRP30 (adipocyte complement-related protein of 30 kDa) (10). It suggested a structural and evolutionary link between the tumor necrosis factor and the gC1q domains and le...

We report the molecular cloning of the human laminin oc3b chain variant and its mRNA expression pattern in adult human tissues when compared to the a3a variant. The mRNA encoding for the o3b variant is about 11 kb and the predicted translation product carries the complete set of domains typical for a 'full-sized' laminin a chain. Apart from the similar domain structure of rx3b also the sequence of rx3 resulted more closely related to the a5 than to the oc4 chain. Quantitative analysis of the RNA expression in a broad panel of adult human tissues indicated that the rx3b variant is more widely distributed than the oc3a shorter variant.

The extracellular matrix glycoprotein EMILIN1 exerts a wide range of functions mainly associated with its gC1q domain. Besides providing functional significance for adhesion and migration, the direct interaction between α4β1 integrin and EMILIN1-gC1q regulates cell proliferation, transducing net anti-proliferative effects. We have previously demonstrated that EMILIN1 degradation by neutrophil elastase (NE) is a specific mechanism leading to the loss of functions disabling its regulatory properties. In this study we further analysed the proteolytic activity of NE, MMP-3, MMP-9, and MT1-MMP on EMILIN1 and found that MMP-3 and MT1-MMP partially cleaved EMILIN1 but without affecting the functional properties associated with the gC1q domain, whereas NE was able to fully impair the interaction of gC1q with the α4β1 integrin by cleaving this domain outside of the E933 integrin binding site. By a site direct mutagenesis approach we mapped the bond between S913 and R914 residues and selected the NE-resistant R914W mutant still able to interact with the α4β1 integrin after NE treatment. Functional studies showed that NE impaired the EMILIN1-α4β1 integrin interaction by cleaving the gC1q domain in a region crucial for its proper structural conformation, paving the way to better understand NE effects on EMILIN1-cell interaction in pathological context.

Electron Beam Melting (EBM) is attracting large interest among the manufacturers of surgical implants as a near-net shape technology. Titanium alloy Ti6Al4V is widely used in the biomedical field thanks to its high biocompatibility, corrosion resistance and mechanical properties. The chemistry and microstructural features of EBM Ti6Al4V indicate lower machinability in comparison with wrought Ti6Al4V. Aim of the paper is to present a comparison between the machinability of wrought and EBM Ti6Al4V in semi-finishing external turning, by quantifying the effects of the cutting speed and the feed rate. Tool wear, surface integrity, chip morphology and microstructural analysis have been used to compare and assess the machinability of Ti6Al4V delivered in the two conditions.