Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model.     

Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis.     

Molecular forms of atrial natriuretic polypeptides circulating in human plasma

Molecular forms of atrial natriuretic polypeptides circulating in human plasma were analyzed by chromatographic separation, coupled with radioimmunoassay. Analyses were done with human plasma samples taken from 24 human subjects, divided into groups of healthy volunteers, patients with renal disease, and patients with heart disease, with 8 subjects in each group. Although alpha-hANP was found as a major circulating form in most of the plasma specimens (16 cases), beta-hANP accompanied by alpha-hANP was found in 6 cases including 2 healthy volunteers. In addition, hANP-immunoreactive macromolecule, which is likely a bound form of hANP, was found to some extent in all the specimens tested. Diversity of human plasma in ANP molecular distribution was discussed in connection with a radioimmunoassay for plasma ANP.     

Isolation and characterization of the circulating form of human endostatin

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C-terminal fragment of collagen α1(XVIII) was guided by MALDI-MS and the exact molecular mass determined by ESI-MS was found to be 18 494 Da. N-terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti-proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.     

The human gastrin precursor. Characterization of phosphorylated forms and fragments.

There is a potential phosphorylation site in the C-terminal region of the precursor for the acid-stimulating hormone gastrin, which is immediately adjacent to an important cleavage point. In the present study we have sought to identify, separate, quantify and characterize phosphorylated and unphosphorylated forms of human progastrin and its fragments. Identification was made by two radioimmunoassays: (a) a novel assay employing an antibody raised to intact human progastrin; and (b) an assay using antibody reacting with the C-terminal tryptic fragment of human progastrin, as well as progastrin itself. Two forms of human progastrin isolated from a gastrinoma were separated by ion-exchange h.p.l.c., and had similar elution positions on reverse-phase h.p.l.c. and on gel filtration. The more acidic peptide contained close to equimolar amounts of phosphate. On trypsinization, peptides were released that co-eluted on ion-exchange h.p.l.c. with, and had the immunochemical properties of, naturally occurring C-terminal fragments of progastrin. One of the latter was isolated and shown by Edman degradation after derivatization with ethanethiol to have the sequence Ser (P)-Ala-Glu-Asp-Glu-Asn. Similar peptides occur in antral mucosa resected from ulcer patients. The unphosphorylated forms of progastrin predominated, whereas the phosphorylated forms of the C-terminal fragments were predominant. This distribution could be explained by preferential cleavage of phosphorylated progastrin. We conclude that in human progastrin, Ser-96 can occur in the phosphorylated form; this residue immediately follows a pair of basic residues (Arg-Arg) that are cleaved during synthesis of the biologically active product.     

Molecular forms of myeloperoxidase in human plasma.

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.     

Ovine atrial natriuretic factor: sequence of circulating forms and metabolism in plasma.

The sequence of ovine ANF is not known, yet sheep have been used extensively for ANF studies. We sequenced the circulating form of ovine ANF from coronary sinus plasma of sheep in paced heart failure. The main circulating form was identical to human ANF(99-126). Small amounts of ANF identical to human ANF(103-126) and ANF(101-126) peptides were also found. Incubation of labeled ANF in ovine serum suggested ANF(103-126) could be a degradation product of ANF(99-126). The endopeptidase-24.11 degradation product ANF(99-105/106-126) was not found in ovine plasma, in contrast to human plasma where it was a minor component. These results show that while the main circulating forms are similar in sheep and humans, there are differences in the minor peptides.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.     

Enhanced adhesion of human mononuclear cells to nonenzymatically glycosylated collagen I

Nonenzymatic glycosylation of extracellular matrix components may contribute to altered interaction of cells with the matrix. We have examined the interaction of mononuclear cells with early glycosylated collagen I. Significantly more cells attached to glycosylated collagen compared to normal collagen. Radioiodinated glycosylated collagen I specifically bound to mononuclear cells in a time and concentration dependent manner with a Kd of 2.45×10^–9 M. Maximum binding was observed in the presence of Mn⁺⁺ ions. The iodinated ligand bound to mononuclear cell membrane immobilized on nitrocellulose disks and the interaction was found to be saturable. These results suggested an alteration in the interaction of human blood mononuclear cells with collagen I, when it gets glycosylated non enzymatically and also indicate that early glycosylated collagen interacts with mononuclear cells through specific, high affinity cell surface molecules. (Mol Cell Biochem148: 115–121, 1995)     

Disproportional release of differently glycosylated forms of human renin by furosemide

Concanavalin A (con A) chromatography of human plasma revealed the presence of three differently glycosylated forms of active renin(AR) and prorenin(PR), including the con A unbound forms(AR-I and PR-I), the loosely-bound forms (AR-II and PR-II), and the tightly-bound forms (AR-III and PR-III). These three forms of AR and PR were observed in human renal extracts. Normal male volunteers were intravenously given the diuretic furosemide (20 mg), kept standing for one hr and the effect on each form of renin was examined. These treatments elevated the plasma concentrations of AR-I, II and III by 2.1 +/- 0.2, 2.6 +/- 0.5, and 6.3 +/- 1.1-fold, respectively (n = 12), thereby indicating that the increase in AR-III was significantly larger than that in the other two forms (P less than 0.01). This disproportional increase was accompanied by a significant increase in the relative percent of AR-III in plasma from 21.6 +/- 2.5 to 42.2 +/- 3.0% (P less than 0.01). On the other hand, increase in the plasma levels of PR-I, II, and III was small (1.4 +/- 0.1, 1.0 +/- 0.1, and 1.1 +/- 0.2-fold, respectively). These results provide evidence for the presence of differently glycosylated forms of AR and PR in human plasma and suggest the preferential release of AR-III with the acute stimulation of renin, by furosemide.     

Fibronectin fragments in human seminal plasma

The study has revealed the presence of fibronectin (FN) fragments and a lack of intact FN in 72 seminal plasma samples. The FN fragmentation was examined by immunoblotting with a monoclonal antibody specific to the central cellular FN domain and was confirmed with a monoclonal antibody directed to the C-terminal domain of FN. Nine FN fragments between 60 and 200 kDa and five fragments of 60-150 kDa were identified in seminal plasma samples of normozoospermic and of terato-, oligoterato-, and oligoasthenoterato-spermic groups, respectively. The relative amounts of the 60, 90 and 100 kDa FN fragments were 2-3 times higher in seminal plasmas with abnormal semen characteristics than in the normozoospermic group. The results suggest that seminal plasma FN fragments may contribute to fertilization and the analysis of FN fragmentation may have a diagnostic value in andrological investigations.     

The multiple forms of alpha-D-mannosidase in human plasma.

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.     

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1)₃; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.Key words: antibody engineering, multivalent antibody, collagen XVIII, collagen XV, tumor targeting     

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1)₃; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.     

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.     

Generation of glycosylated remnant epitopes from human collagen type II by gelatinase B

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is an inflammatory mediator and effector. Considerable amounts of gelatinase B are released by neutrophils in the synovial cavity of patients with rheumatoid arthritis, and gelatinase B-deficient mice are resistant against antibody-induced arthritis. Native human collagen type II is susceptible to cleavage by various collagenases (MMP-1, MMP-8, and MMP-13), which cleave at a single position in the triple helix. Although the triple-helical structure may persist after this single cleavage, we show that gelatinase B degrades the resulting fragments into small remnant peptides. These were identified by mass spectrometry and Edman degradation. Localization of 31 cleavage sites shows that the immunodominant epitopes remain intact after cleavage and may become available, processed as antigens and presented in MHC-II molecules. Furthermore, most post-translational modifications were identified on the fragments, including nine glycosylation sites. In particular, it is shown for the first time by structural analysis that in natural human collagen II, lysines in the main immunodominant epitope are modified by partial hydroxylation and partial glycosylation. Determination of T-cell reactivity against such fragments indicates that, besides the two known main immunodominant epitopes, other glyco-epitopes may be present in collagen II. This reinforces the role of glycopeptide antigens in autoimmunity.     

Proteoglycan-collagen XV in human tissues is seen linking banded collagen fibers subjacent to the basement membrane.

Type XV is a large collagen-proteoglycan found in all human tissues examined. By light microscopy it was localized to most epithelial and all nerve, muscle, fat and endothelial basement membrane zones except for the glomerular capillaries or hepatic/splenic sinusoids. This widespread distribution suggested that type XV may be a discrete structural component that acts to adhere basement membrane to the underlying connective tissue. To address these issues, immunogold ultrastructural analysis of type XV collagen in human kidney, placenta, and colon was conducted. Surprisingly, type XV was found almost exclusively associated with the fibrillar collagen network in very close proximity to the basement membrane. Type XV exhibited a focal appearance directly on the surface of, or extending from, the fibers in a linear or clustered array. The most common single arrangement was a bridge of type XV gold particles linking thick-banded fibers. The function of type XV in this restricted microenvironment is expected to have an intrinsic dependence upon its modification with glycosaminoglycan chains. Present biochemical characterization showed that the type XV core protein in vivo carries chains of chondroitin/dermatan sulfate alone, or chondroitin/dermatan sulfate together with heparan sulfate in a differential ratio. Thus, type XV collagen may serve as a structural organizer to maintain a porous meshwork subjacent to the basement membrane, and in this domain may play a key role in signal transduction pathways.