The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and em- bryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFR), and disturbed the TNFα/TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.

A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are consistent with a chain length dependent cooperative folding of the peptides into coiled coils.

A new member of the human cystatin superfamily, called cystatin E, has been found by expressed sequence tag (EST) sequencing in amniotic cell and fetal skin epithelial cell cDNA libraries. The sequence of a fulllength amniotic cell cDNA clone contained an open reading frame encoding a putative 28-residue signal peptide and a mature protein of 121 amino acids, including four cysteine residues and motifs of importance for the inhibitory activity of Family 2 cystatins like cystatin C. Recombinant cystatin E was produced in a baculovirus expression system and isolated. An antiserum against the recombinant protein could be used for affinity purification of cystatin E from human urine, as confirmed by N-terminal sequencing. The mature recombinant protein processed by insect cells started at amino acid 4 (cystatin C numbering), and displayed reversible inhibition of papain and cathepsin B (K i values of 0.39 and 32 nM, respectively), in competition with substrate. Cystatin E is thus a functional cysteine proteinase inhibitor despite relatively low amino acid sequence similarities with human cystatins (26 -34% identity with sequences for the Family 2 cystatins C, D, S, SN, and SA; <30% with the Family 1 cystatins, A and B, and domains 2 and 3 of the Family 3 cystatin, kininogen). Unlike other human low M r cystatins, cystatin E is a glycoprotein, carrying an N-linked carbohydrate chain at position 108. Northern blot analysis revealed that the cystatin E gene is expressed in most human tissues, with the highest mRNA amounts found in uterus and liver. A strikingly high incidence of cystatin E clones in cDNA libraries from fetal skin epithelium and amniotic membrane cells (>0.5% of clones sequenced) indicates a protective role of cystatin E during fetal development.

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