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PubMed2003

Proteome analysis of conditioned medium from cultured adult hippocampal progenitors.

It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo.

Aging↗

PubMed1997

The cysteine proteinase gene cprG in Dictyostelium discoideum has a serine-rich domain that contains GlcNAc-1-P.

A lysosomal cysteine proteinase called proteinase-1 is the major proteolytic enzyme in vegetative cells of Dictyostelium discoideum. This phosphoglycosylated protein contains multiple residues of GlcNAc-1-P linked to peptidyl serines. Here we report the cloning, structure, and expression of its cDNA (cprG). Another cDNA (cprF) closely related to cprG was also cloned and characterized. mRNAs of both genes are present during the vegetative phase and decrease in developing cells. However, the level of cprG mRNA is about 100-fold higher than that of cprF. The predicted protein products of both genes contain a unique serine-rich domain that was previously found only in two Dictyostelium proteinases (CP4 and CP5) that also carry a GlcNAc-1-P-Ser modification. The cprG product, renamed CP7, was tagged with the FLAG-epitope (FLAG-CP7) and shown to bind to cystatin, a highly specific inhibitor of cysteine proteinases. The FLAG-CP7 product also contained both N-linked oligosaccharides and GlcNAc-1-P. Deletion of the serine-rich domain from FLAG-CP7 yields a product that still binds to cystatin, but no longer carries GlcNAc-1-P. This finding supports the idea that the GlcNAc-1-P residues are normally added to the serine-rich domain, found only in vegetative Dictyostelium cysteine proteinases.

Acetylglucosamine↗

PubMed1995

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat.

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6) but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.

Androgens↗

PubMed1987

Separation and partial characterization of four cysteine proteinases from a human epidermal cell line.

Four different cysteine proteinases from a cultured human epidermal cell line (NCTC 2544) were partially purified and characterized. The biggest hydrolase was an endoaminopeptidase with the molecular weight of several hundred kilodaltons. It was a glycoprotein and had an almost neutral pH optimum. The three other hydrolases resembled lysosomal cathepsins B, H, and L in various respects except for somewhat higher molecular weight for cathepsin B (29 kDa) and the cathepsin H-like (70 kDa) hydrolase than those reported from most other tissues. Low molecular weight cysteine proteinase inhibitors ACPI (cystatin A) and NCPI (cystatin B) inhibited the cathepsins, but not the high molecular weight proteinase.

Cell Line↗

PubMed1999

Bunina bodies in amyotrophic lateral sclerosis on Guam: a histochemical, immunohistochemical and ultrastructural investigation.

An investigation of Bunina bodies is important when studying the pathoetiology and pathomechanisms involved in amyotrophic lateral sclerosis (ALS). It may serve as a clue essential for the study of the pathogenesis of Guamanian amyotrophic lateral sclerosis (ALS-G), and it may provide a means of answering the question of whether ALS-G is the same disease as classical ALS or a different entity. In ALS-G, however, no precise histochemical, immunohistochemical, or detailed ultrastructural examination has been published to date. To elucidate the pathological differences/similarities of Bunina bodies between classical ALS and ALS-G, we performed histochemical, immunohistochemical, topographic and ultrastructural examinations. Histochemically, hematoxylin and eosin, Masson's trichrome, methylgreen-pyronin, phosphotungstic acid-hematoxylin, Klüver-Barrera, Bodian and periodic acid-Schiff staining were utilized. Immunohistochemical examination was performed using antibodies for cystatin C, ubiquitin, Tau-2, Cu/Zn superoxide dismutase, phosphorylated neurofilament and glial fibrillary acidic protein. Histochemical findings were consistent with those previously described for classical ALS. The immunohistochemical study showed that in ALS-G Bunina bodies were intensely labeled by an anti-cystatin C antibody. Topographic examination demonstrated that Bunina bodies were distributed in the spinal anterior horns and Clarke's column in the spinal cord. Ultrastructurally, Bunina bodies were composed of electron-dense amorphous/ granular material accompanied by vesicular structures and neurofilaments. The results of the present study have revealed that the pathological features of Bunina bodies in ALS-G are identical to those seen in classical ALS. These findings strongly suggest that a similar degenerative process occurs in the spinal anterior horn cells in both ALS-G and classical ALS.

Adult↗

PubMed2002

Correct evaluation of renal glomerular filtration rate requires clearance assays.

Iohexol clearance is an accepted, but time-consuming assay for the measurement of glomerular filtration rate (GFR). We investigated if simpler methods could predict GFR. Sixty-nine children with hematological-oncological disorders participated. A linear relationship was established by regression analysis between iohexol clearance ( n=734) and 1/s-creatinine ( r=0.45, n=727), s-cystatin C ( r=0.41, n=518), and the Schwartz ( r=0.45, n=723), Counahan-Barratt ( r=0.48, n=723), and modified Counahan-Barratt formulae ( r=0.48, n=723). These correlations improved when one GFR measurement per individual was compared with each of the five parameters. We further investigated if iohexol clearance could accurately be replaced. The degree of variation in predicting GFR was estimated by the standard deviation of the residuals (S(res)). For 1/s-creatinine and s-cystatin C, S(res) was 39 and 38 ml/min per 1.73 m(2). For the formulae of Schwartz, Counahan-Barratt, and modified Counahan-Barratt, the S(res) was 43, 40, and 40 ml/min per 1.73 m(2), respectively. The wide variations of the S(res) were not reduced when one GFR measurement per child was compared with the five parameters. Due to the large deviation in predicting GFR, we conclude that the five alternative methods studied cannot replace iohexol clearance for measurement of GFR.

Adolescent↗

PubMed2007

Glomerular filtration rate measurement and estimation in chronic kidney disease.

Glomerular filtration rate (GFR) assesses kidney function. GFR is measured by renal clearance techniques; inulin clearance is the gold standard but is not easily measured. Thus, other methods to determine GFR have been utilized. Endogenous creatinine clearance (CrCl) is the most widely used, but creatinine secretion falsely elevates GFR. Cimetidine inhibits creatinine secretion, such that CrCl equals GFR, provided there are no difficulties with bladder emptying. Estimation of GFR from serum creatinine (e.g. Schwartz formula) is useful clinically; however, such formulae have not been updated for enzymatic creatinine autoanalyzers. Cystatin C, a small protein, is produced at a relatively constant rate and is reabsorbed in the proximal tubule. Cystatin C may be more sensitive than creatinine in detecting a reduction in GFR, but further studies are needed to prove this. Single injection (plasma) clearance techniques are the most precise measures of GFR. Iohexol is an exogenous marker that is comparable to inulin and (51)Cr-EDTA and can be measured by high-performance liquid chromatography (HPLC). Our pilot and the Chronic Kidney Disease in Children (CKiD) North American studies show that iohexol can accurately measure GFR using a four-point plasma disappearance curve national studies show that iohexol can accurately measure GFR using a four-point plasma disappearance curve (10, 30, 120, and 300 min) or, in most cases, a two-point disappearance time (120 and 300 min).

Education, Medical, Continuing↗

PubMed1993

Participation of cathepsin L on bone resorption.

The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Animals↗

PubMed1993

Cloning, expression and characterization of human kininogen domain 3.

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM-[G253-S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined Ki values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (Ki = 63 nM) as it has been found for native L-kininogen (Ki = 340 nM).

Amino Acid Sequence↗

PubMed1994

Lack of expression of T-kininogen gene in the hearts of untreated and turpentine-injected rats.

Cystatins, inhibitors of cysteine proteinases, are present in rat heart. However, the controls of genes coding for various cystatins in the heart, and the cellular sites of expression of these genes are not known. With a sensitive reverse transcriptase--polymerase chain reaction T-kininogen mRNA was readily detected in the submandibular glands and livers, but not in the hearts, of control or turpentine-injected rats. Immunocytochemical observations employing a monoclonal antibody to bradykinin, which reacts with kininogens in general, revealed no specific staining in cardiac structures, but a weak staining was apparent in blood vessels and on the surface of endothelial cells of both control and turpentine-injected rats. The monoclonal antibody revealed the presence of kininogens in the acinar cells of the submandibular gland, and, in acute inflammation, in the hepatocytes. These findings suggest that the T-kininogen gene is not expressed in the heart, and the T-kininogen demonstrable in heart extracts derives from the blood. Circulating kininogens are likely bound to endothelial cells, and may be a local source of kinins. In addition, kininogens, as potent inhibitors of cysteine proteinases, may play a role in pathologic conditions of the heart by controlling the deleterious effects of cathepsins released from lysosomes or secreted by macrophages.

Animals↗

PubMed1994

Mechanism and regulation of antigen processing by cathepsin B.

Cellular and humoral immune responses to vaccines of hepatitis B and rabies as antigens were suppressed by specific inhibitors of cathepsin B, anti-cathepsin B antibody and the specific substrate of cathepsin B. The antigenic peptides of these vaccines are processed by cathepsin B and the fragments are capable of binding with the desetope of MHC class II, beta-chain, because one of the active sites of cathepsin B (14, 15) VN217-222 shares high homology with a part of the desetope, VN57-62, of MHC class II, beta-chain. Rechallenge of the synthesized antigenic peptides of these vaccine molecules shows a strong proliferative response to the splenocyte primed by these vaccines. However, the response to these antigenic peptides was not inhibited by cathepsin B inhibitors. These findings suggest that cathepsin B inhibitors do not inhibit any other processes of immune responses than the proteolytic processing of antigens. Some investigators reported recently that the Ii-chain is degraded by purified cathepsin B in vitro (23-25). However, we showed that the suppression of these immune responses by cathepsin B inhibitors is not due to the inhibition of invariant chain degradation. We found that the invariant chain shares about 40% homology with the cystatin family which are the endogenous inhibitors of cysteine proteases (23, 24). Therefore, the Ii-chain is one of the members of the cystatin superfamily and may participate in the regulation of presentation of antigenic peptides and also antigen processing by cathepsin B.

Amino Acid Sequence↗

PubMed1992

Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.

Animals↗

PubMed1992

Comparison between the Icelandic and Dutch forms of hereditary cerebral amyloid angiopathy.

Hereditary cerebral amyloid angiopathy has been described in Icelandic and Dutch families. Although the clinical manifestations show similarities, biochemical characterization revealed that amyloid in the Icelandic patients consists of cystatin C and in the Dutch patients of beta-protein. Both diseases are caused by a single base mutation leading to the same amino acid (viz. glutamine). Furthermore, both cystatin C and the beta-protein precursor are protease inhibitors. Therefore, the mechanism of amyloidogenesis may be similar in both diseases. A comparison of clinical, pathological, genetic, and biochemical aspects of these two types of hereditary cerebral amyloid angiopathy is presented.

Amyloid beta-Protein Precursor↗

PubMed2026

Proteomic characterization of the acquired enamel pellicle under acidic challenges at early and mature formation stages.

OBJECTIVES: This study aimed to characterize acquired enamel pellicle (AEP) proteomic changes after exposure to citric acid (CA) and hydrochloric acid (HCl) under different pellicle formation times (3 and 120 min) in the same volunteers. DESIGN: Nine healthy volunteers participated in this randomized crossover in vivo study. The AEP was allowed to form for 3 or 120 min and subsequently exposed for 10 s to deionized water (control), 1% CA (pH 2.5), or 0.01 M HCl (pH 2.0). Pellicle samples were collected, followed by protein extraction, tryptic digestion, and analysis by nanoliquid chromatography (nanoLC) coupled to mass spectrometry (MS) with MSE (data-independent acquisition; nanoLC-MSᴱ). Label-free quantitative proteomics were performed for relative quantification using t-test (p < 0.05). RESULTS: At 120 min, CA exposure markedly reduced several typical AEP proteins, especially acidic proline-rich proteins (PRPs). Conversely, basic PRPs were upregulated, suggesting acid-resistance protein signature. At 3 min, basal-layer proteins (PRPs, cystatins, histatins and mucins) were more abundant. Hemoglobins increased 6-8-fold (up to 150-fold in 3 min control), suggesting association with early pellicle formation and an acid-resistant protein signature. CA exposures for 120 min also upregulated typical AEP proteins (PRPs, mucins, cystatins, immunoglobulins), while HCl exposure depleted albumins and lactotransferrin. CONCLUSION: Intrinsic and extrinsic acids induce distinct proteomic signatures in the AEP. Hemoglobin and PRPs appear consistently enriched in the early pellicle layer, reflecting an initial acid-resistant protein signature. These findings provide new insights into the molecular remodeling of the AEP following intrinsic and extrinsic acid exposure, highlighting proteins potentially involved in early-stage pellicle formation.

Humans↗

PubMed2006

Identification and expression of gene transcripts generated during an anti-parasitic response in Biomphalaria glabrata.

In order to gain further insights into the molecular basis of gastropod anti-parasite immune responses, we investigated transcripts of Biomphalaria glabrata regulated during hemocytic encapsulation. Using a snail strain that is resistant to the parasite Echinostoma caproni, we performed suppression subtractive hybridization (SSH) to construct cDNA libraries of transcripts more abundantly expressed in unexposed or parasite-exposed snails. After sequence analysis and quantitative PCR analysis of expression, we identified 10 candidates of particular interest. They belonged to various functional groups such as detoxification enzymes (GST, SOD), antimicrobial proteins (LBI/BPI), protease inhibitors (cystatins), calcium-binding proteins, or C-type lectins. In situ hybridization (ISH) analysis revealed that one overexpressed cystatin-like candidate is specifically expressed in hemocytes participating in parasite encapsulation or aggregating at the site of infection. Two other candidates (C-type lectin and a LBP/BPI) were expressed in the albumen gland, further supporting the role of this organ in immunity and/or host-parasite interaction.

Animals↗

PubMed2004

Renal recovery after conversion to a calcineurin inhibitor-free immunosuppression in late cardiac transplant recipients.

OBJECTIVE: Calcineurin inhibitor (CNI)-related renal failure is a common problem after cardiac transplantation (HTx). The aim of this prospective study was to evaluate the safety and efficacy of a completely CNI-free immunosuppressive regimen [mycophenolate mofetil (MMF) and sirolimus (Sir)] in HTx-recipients with late post-transplant renal impairment. METHODS: Since 2001, 30 HTx-patients (25 men, 6 women; 0.2-14.2 years after transplantation) with CNI-based immunosuppression and a serum creatinine >1.9 mg/dl were included in the study. Creatinine and cystatin levels were monitored to detect renal function. Conversion was started with 6 mg Sir or 500 mg MMF according to the pre-existing regimen and was continued with the dose adjusted to achieve target trough levels between 8 and 14 ng/ml (Sir) or 1.5 and 4 microg/ml (mycophenolate). Subsequently, the CNIs were tapered down and stopped. Clinical follow-up included endomyocardial biopsies, echocardiography and laboratory studies. Additionally, every HTx-patient treated at our centre between 1996 and 2001 due to chronic renal failure without immunosuppressive conversion and fulfilling the inclusion criteria were retrospectively analysed and acted as control group. RESULTS: Patient demographics and 1-year survival [93 (conversion) vs 90% (control)] were compared. No acute rejection episode was detected in either group. Renal function improved significantly in the conversion group (creatinine: 3.18+/-0.71 vs 2.22+/-0.79 mg/dl, P=0.001; cystatin pre- vs post-conversion: 2.95+/-1.06 vs 2.02+/-1.1 mg/l, P=0.01). In three patients haemodialysis therapy was stopped completely after conversion. In the control group renal impairment was deteriorating, creatinine increased from 2.44+/-0.8 to 3.28+/-1 mg/dl (P=0.01). In 10 out of 33 patients chronic haemodialysis had to be initiated within 1 year. Although side effects of CNI-free immunosuppression were common (76%), no patient had to be excluded due to adverse effects. CONCLUSIONS: Conversion from CNI-based immunosuppression to MMF and Sir in HTx-patients with chronic renal failure was safe, preserved graft function and improved renal function.

Blood Pressure↗

PubMed2025

Polygenic Risk Scores Predicting Estimated GFR Validated With Iohexol Clearance.

INTRODUCTION: Genome-wide association studies (GWAS) have identified hundreds of single nucleotide variants (SNVs) associated with estimated glomerular filtration rate (eGFR). eGFR has been used as a proxy phenotype because of the complexity and cost of measured GFR (mGFR) in large studies. Because eGFR is influenced by non-GFR factors, these GWAS results may be biased compared with a hypothetical study using mGFR. We aimed to investigate this by comparing aggregate measures of genetic effects on mGFR and eGFR. METHODS: We studied 1492 persons from the Renal Iohexol Clearance Survey (RENIS) cohort, a representative sample of the general population in Northern Norway without preexisting cardiovascular disease, kidney disease, or diabetes. We measured iohexol-clearance, and genotyping was performed with a microarray chip enriched for GFR-related SNVs. We compared the performance of 3 published polygenic risk scores (PGS) developed for creatinine-based eGFR (eGFRcr), narrow-sense heritability (h2) and the mean effect of SNVs on mGFR, eGFRcr, cystatin C-based eGFR (eGFRcys) and eGFRcr-cys. RESULTS: The performance of the PGS differed for mGFR and the 3 eGFRs, with best performance for prediction of eGFRcr (P < 0.05). However, when the beta coefficients of the SNVs in the 3 PGS were estimated in the RENIS-cohort, their magnitude was 11% to 46% greater for mGFR than for the 3 eGFR methods in 8 of 9 comparisons (P < 0.05). mGFR had higher h2 (0.47) than eGFRcr (0.21), eGFRcys (0.37), and eGFRcr-cys (0.42). CONCLUSIONS: SNVs with non-GFR effects on creatinine and cystatin-C influence GWAS results. The results of GWAS using eGFR should be validated using experimental and other more precise methods.

chronic kidney disease↗

PubMed2005

BMP binding peptide: a BMP-2 enhancing factor deduced from the sequence of native bovine bone morphogenetic protein/non-collagenous protein.

Forty years ago, Marshall Urist described a partially purified extract of demineralized bone matrix which induced the formation of ectopic bone. This substance, bone morphogenetic protein/non-collagenous protein (BMP/NCP), was never purified to homogeneity but other investigators used similar starting materials to clone a number of recombinant BMPs. Urist recognized that his material probably contained the BMPs which had been cloned by others but always contended that it contained another, more potent, bone inducing material which differed significantly in its physical and chemical properties from the known BMPs. We have used Urist's protocol to isolate a protein that has the chemical and physical properties of Urist's "BMP". It is an 18.5 kD fragment of the bone matrix protein, SPP-24. This fragment contains the cystatin-like domain of SPP-24. We have located a 19 amino acid region which is similar to the TGF-beta/BMP-binding region of fetuin, a member of the cystatin family of protease inhibitors. A cyclic peptide, which we call BMP binding peptide (BBP) was generated using this sequence. The peptide avidly bound rhBMP-2 with a KD of 3 x 10(-5) M. When implanted alone in mouse muscle, the peptide frequently induced dystrophic calcification. When implanted with rhBMP-2, the peptide enhanced the osteogenic activity of the recombinant molecule. We hypothesize that Urist's "BMP" was a fragment of SPP-24 which influenced bone induction by binding to bone morphogenetic proteins. BBP may be clinically useful because of its effects on other bone-inducing substances.

Amino Acid Sequence↗