Scanning Microscopy

Volume 10 Number 2 Article 11

11-22-1995

Inter-Alpha-Inhibitor: A Protein Family Involved in the Inhibition of Calcium Oxalate Crystallization

Fouad Atmani University of Florida College of Medicine

Jacques Mizon Universite de Lille

Saeed R. Khan University of Florida College of Medicine

Follow this and additional works at: https://digitalcommons.usu.edu/microscopy

Part of the Biology Commons

Recommended Citation Atmani, Fouad; Mizon, Jacques; and Khan, Saeed R. (1995) "Inter-Alpha-Inhibitor: A Protein Family Involved in the Inhibition of Calcium Oxalate Crystallization," Scanning Microscopy: Vol. 10 : No. 2 , Article 11. Available at: https://digitalcommons.usu.edu/microscopy/vol10/iss2/11

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 10, No. 2, 1996 (Pages 425-434) 0891-7035/96$5.00+.25 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA INTER-ALPHA-INIIlBITOR: A PROTEIN FAMILY INVOLVED IN THE INHIBITION OF CALCIUM OXALATE CRYSTALLIZATION

Fouad Atmani1, Jacques Mizon2, and Saeed R. K.han1••

1Univ. Florida College of Medicine, Dept. Pathology and Laboratory Medicine, Gainesville, FL 32610-0275, USA 2Universite de Lille, Faculte de Phannacie, Laboratoire de Biochimie, 59006 Lille Cedex, France (Received for publication April 23, 1995 and in revised form November 22, 1995)

Abstract Introduction

Inter-a-inhibitor (Jal) is a serine protease inhibitor Mammalian blood is a rich source of proteinase in­ present in human plasma. It has a molecular weight of hibitors which account for about 10 % by weight of all about 220 kDa which encompasses 3 chains including plasma proteins in human [46 , 69]. They constitute the two heavy chains and one light chain. The light chain, third largest group of functional proteins after albumin known as bikunin, is responsible for the antitryptic ac­ and immunoglobulins. Inter-a-inhibitor (Jal) is one tivity of Jal in the inhibition of various enzymes, such among the well characterized human serine plasma pro­ as trypsin and chymotrypsin. Under physiologic or cer­ teinase inhibitors. These inhibitors seem to control di­ tain pathologic circumstances, several macromolecules verse critical events associated with proteolysis that oc­ related to Jal appear in plasma and urine. However, the cur during pathologic circumstances such as cancer, fi­ physiologic role of Jal remains unclear. As far as uro­ brinolysis, and inflammation by inhibiting the action of lithiasis is concerned, two urinary macromolecules relat­ various enzymes including neutrophil elastase, cathepsin ed to Jal have been isolated and shown to be potent in­ G, and plasmin [20, 28, 50] . Nevertheless, the physio­ hibitors of calcium oxalate formation. One of these in­ logical function of fol is still not well established. The hibitors, uronic-acid-rich protein (UAP), has been iden­ average concentration of fol in plasma of healthy human tified and well characterized. The sequence of the first subjects is about 450 mg/I [65]. Low amounts of other 18 amino acid residues of UAP is identical with that of smaller macromolecules immunologically related to Jal bikunin. Furthermore, the immunoreaction between are also present in plasma and urine. Their level in­ UAP and Jal antibody using immunoblot analysis was creases in pathological conditions, suggesting that they positive. UAP isolated from the urine of stone formers could be a result of an increase of Jal turnover. Many exhibited less inhibitory activity towards calcium oxalate of these macromolecules have been purified and charac­ crystalliz.ation than that derived from the urine of healthy terized to understand the mechanism of their release and subjects. This suggests a structural abnormality of the the importance of their presence in the body fluids. A inhibitor obtained from stone patients. The organic few years ago, two macromolecules related to fol and matrix extracted from kidney stones contained a protein having the ability to inhibit calcium oxalate (CaOx) crys­ antigenically related to Jal. We conclude that UAP is talliz.ation in vitro were isolated from human urine [2, a member of Jal family taking part in inhibiting calcium 63] . oxalate crystalliz.ation, and modulating the formation of This paper is written to focus attention on the rela­ stones in the urinary tract. tionship between Jal and urolithiasis. We describe here Jal and its related proteins, specifically those that may Key Words: Urolithiasis, urine, plasma, calcium oxa­ be involved in stone formation. late, inter-a-inhibitor, uronic-acid-rich protein, bikunin. Structure of fol and its Immunologically • Address for Correspondence: Related Derivatives (Figure 1) Saeed R. Khan University of Florida, College of Medicine, Jal is a glycoprotein with a molecular weight of Department of Pathology and Laboratory Medicine, about 180-220 kDa. A decade ago, it was thought that Gainesville, FL 32610--0275, USA. Jal contained a single polypeptide chain since it showed Telephone number: (352) 392-3574 a unique band on sodium dodecyl sulfate polyacrylamide FAX number: (352) 392-6249 gel electrophoresis (SDS-PAGE) in the presence of E. mail: [email protected] strong reducing agents [41, 51]. However, subsequent

425 F. Atmani, J. Miron and S.R. Khan

a,1-m/Bikunin precursor HI H2 H3 ISSS SSSSSS SSSSSSSS SSS SSS 1 I= :::: ======::==: ::: :::::::: ::: =::==== I 78 kDa 85 kDa 85 kDa

a,1-m Free bikunin G••·••·••••·w.. w.l 31 kDa / 45 kDa GAG ~

Ia,I H2/Bikunin Pal 220 kDa 130 kDa 130 kDa

Figure 1. Structure oflal and its derivatives according to reference 54. Bikunin is derived from a common precursor that codes also for al-microglobulin (al-m). It is present in a free form or complexed with one or two heavy (H) chains constituting H2/Bikunin, inter-a-inhibitor (Ial) (Hl + H2 + bikunin), or pre-a-inhibitor (Pal) (H3 + bikunin). A glycosaminoglycan (GAG) chain, associated with bikunin, ensures the junction between the different subunits of lal and its related proteins. ------data coming from molecular biology techniques demon­ Bikunin is also present in a free form in plasma and strated the presence of two or three distinct mRNAs cod­ urine [54]. Interestingly, the three chains are held to­ ing for heavy (H) and light (L) chain respectively [11, gether in unique complex by way of a carbohydrate 19, 25, 56, 60]. The latter is called bikunin, formerly chain [32, 38]. Bikunin contains a N-linked oligosaccha­ known as urinary trypsin inhibitor, acid-stable trypsin ride chain and an O-linked oligosaccharide chain at­ inhibitor, the light chain of lal, or HI-30. Bikunin tached to Asn45 and Ser10 respectively [25, 29]. The originates from a common mRNA that also codes for an­ latter was further demonstrated to be a glycosaminogly­ other protein called al-microglobulin (al-m) [34, 37, can (GAG) [17]. Several studies demonstrated that 53, 62, 71]. As soon as they are synthesized, a cleav­ chondroitinase and hyaluronidase can split lal into heavy age occurs and both al-m and bikunin are released sepa­ and light chains [8, 9, 61]. Recent results confirmed rately [49]. al-m has never been found associated with that the GAG chain covalently cross-linked the subunits Ial or its related polypeptides [56, 60]. In humans, the oflal [40]. scission seems to occur at the amino acid sequence Arg­ Another protein related to lal has also been found Val-Arg-Arg immediately preceding the bikunin subunit and named pre-a-inhibitor (Pal) [22]. It is made of bi­ of the precursor [13]. Therefore, these amino acid resi­ kunin, similar to that of Ial and a heavy chain (H3: 85 dues are missing in the final product. Yet, the mecha­ kDa) coded by another mRNA different from those de­ nism and conditions under which the precursor is broken scribed earlier for Ial. This demonstrated that Pal con­ away remain elusive. Now, it is recognized that Ial stituted a protein distinct from Ial and not derived from comprises two heavy chains (Hl: 78 kDa and H2: 85 it by proteolysis [22]. A protein consisting of H2 and kDa) and bikunin (45 kDa) [10, 38, 45, 52]. The genes bikunin is also present in the plasma [18, 21, 54]. The of these chains are located on the chromosome 3p21.1- nature of the cross-linking between the two polypeptide p21. 2, 10p14-pl5, and 9q32-q34 respectively [19, 59]. chains of this protein appears similar to that found in

426 Inter-a-inhibitor in urolithiasis

Pal. In all polypeptide complexes, Ial, H2/bikunin, or Table l. Distribution of Ial and its derivatives in Pal, bikunin seems to be responsible for their antitryptic normal tissues. activity by inhibiting various enzymes, particularly tryp­ sin and chymotrypsin [20, 28, 50). Type of Method References When Ial and bikunin are submitted to a prolonged tissue digestion with trypsin, they release fragments of a mo­ lecular weight of 14 and 8 kDa named HI-14 and Hl-8, Liver Immunohistology [15) respectively [30, 72). The amino acid composition of mRNA analysis [37·, 55, 56, 67.) bikunin and Hl-14 are identical indicating that HI-14 is Lung Elisa [15) the peptidic chain of bikunin and large carbohydrate Immunohistology [73) moieties should be responsible for the molecular weight of 45 kDa of the native form of bikunin. Therefore, HI- Kidney Immunohistology [15, 44, 73, 74] 14 and HI-8 may be products of Ial or bikunin which Brain Immunohistology [15, 73, 74] are fragmented during purification or in the presence of Stomach lmmunohistology [73, 74] enzymes like trypsin. mRNA analysis [67.] Testis lmmunohistology [15] Biosynthesis and Localization of fol and its Derivatives (Table 1) •Distribution of a 1-microglobulin/Bikunin mRN A Ial is synthesized mainly in the liver and excreted ------in plasma [10, 62). This finding was confirmed at the however, these roles have not been resolved. IaI is es­ molecular level. Indeed, the mRNA encoding for the pecially sensitive to some enzymes such as elastase and Ial subunits was found expressed only in the liver [56). cathepsin G which are activated during several diseases Nevertheless, the expression of the gene encoding for including cancer, inflammation, and renal failure. Due the heavy chain H3 has also been observed in the brain to their smaller size, proteins obtained from Ial turnover [55]. By using dot blot analysis, the mRNA encoding may be able to diffuse throughout the affected tissues for the precursor a 1-microglobulin/bikunin was found in and may contribute to the cells' protection against pro­ rat at high level in the liver and kidney, low in the brain teolysis. Nevertheless, Ial accounts for less than 5% of and testis [33). Further studies by the same authors the total trypsin inhibitory activity [6, 57] which does however, confirmed its synthesis in the liver only [37). not favor such a protective role. Moreover, the results In pig, the expression of mRNA of this precursor was showed no significant consumption of Ial during the in­ observed at high level in the liver and, at a low level, in flammation process, although proteins related to IaI the stomach [67). were present [58]. However, these results were con­ The immunochemical distribution of Ial and its de­ tested in other studies [26, 45]. rivative fragments in different organs has been investi­ McKeehan et al. [39] isolated from human hepato­ gated (Table 1). Under normal conditions, Ial immuno­ ma cells two endothelial cell growth factors structurally reactivity was found in the liver, kidney, testis, gross similar to pancreatic secretory trypsin inhibitor and intestine, cutis and brain [15, 73, 74). Surprisingly, the bikunin. Both proteins are absent or present in low immunoreaction was also detected in cultured fibroblasts amounts in normal plasma and urine. They stimulate [14) and human mast cells [44). Also using al-micro­ endothelial cell growth and their concentrations were globulin antibody, a positive immunoreaction was ob­ increased during inflammation and cancer. These find­ served in human lymphocytes and macrophages [33). ings suggest that Ial and their related proteins may be The presence of Ial related proteins in the urinary tract involved in neovascularii.ation and desmoplasia, associ­ has been specially investigated [44). Ial immunoreac­ ated with tumor and tissue damage process. tivity was found to be exclusively present in renal proxi­ Lastly, S0rensens work [63] and particularly our mal tubules due to the reabsorption of bikunin by this studies [2] demonstrated that a glycoprotein related to segment of nephron. Ial immunoreactivity was also de­ Ial, most probably a urinary bikunin, is a potent inhibi­ tected in human mast cells in the connective tissue of a tor of CaOx crystal growth in vitro and may inhibit and bladder papilloma and in the rat bladder epithelium [ 44). reduce the formation of CaOx crystals in urinary tract.

Physiological Role of fol and its Related Proteins Relationship of fol and its Derivatives to Urolithiasis

The presence of Ial in various tissues suggests that S0rensen et al. [63] isolated a protein from normal it may fulfill different physiological functions. So far, human urine using two anion exchange chromatography

427 F. Atmani, J. Mizon and S.R. Khan

Table 2. Comparison of physico-chemical properties of S0rensens protein, uronic-acid-rich protein (UAP), and urinary bikunin.

Proteins Molecular CaOx growth Effect of pronase on Effect of chondroitinase weight (kDa) inhibitory activity CaOx inhibition

Sorensen's protein 40 +++ No effect U ronic acid removed UAP 35 +++ Activity destroyed Produces a protein of 20 kDa Urinary bikunin 45 + Activity destroyed Produces a protein of 20 kDa steps (on DEAE-Sephacel and Mono Q column) and one and after treatment with pronase, chondroitinase AC, affinity chromatography (vinylsulfone agarose). The and hyaluronidase as described by S0rensen et al. [63]. protein exhibited quite the same characteristics as uri­ Our data demonstrated that chondroitinase and hyalu­ nary bikunin: molecular weight in SDS-PAGE: 40 kDa, ronidase had no effect on CaOx growth inhibitory ac­ in gel filtration: 67 kDa, identical N-terminal sequence, tivity of UAP, but yielded a protein with a molecular and presence of uronic-acids. The authors found that weight of 20 kDa [4]. This finding suggests that UAP this Icd fragment was a potent inhibitor of CaOx crystai comprises a large giycosaminoglycan chain responsible growth and stated that it is the only protein which might for its molecular weight at 35 kDa. In contrast to have an inhibitory activity in the urine. By chondroi­ S0rensens study, pronase treatment degraded the total tinase digestion, the uronic acids were removed but the inhibitory activity of UAP suggesting that the inhibitor inhibitory capacity of the protein remained. Moreover, isolated by us is different from the one purified by pronase digestion had no effect upon its inhibitory capac­ S0rensen et al. [63]. A comparison of physico-chemical ity and the authors considered that this data might be ex­ proprieties of these proteins is shown in Table 2. We plained by the antiprotease activity of the inhibitor [63]. hypothesize that both UAP and S0rensens protein may Unfortunately, further characterization of this inhibitor exist in urine as separate entities or they may be the has so far not been reported. same protein purified by slightly different methods. In an attempt to find another CaOx inhibitor, we fractionated human urinary macromolecules using three Is UAP Bikunin? chromatographic procedures including DEAE-Sephacel gel followed by Sephacryl S-300 chromatography, and The results of amino acid sequence and immunoblot­ finally Mono Q column using an FPLC system. We ting analysis suggest that UAP is a protein related to fol were able to isolate and purify a macromolecule with a family and could be bikunin. To clarify this relation­ molecular weight of 35 kDa as estimated by SDS-P AGE ship, in our recent study (Atmani et al., Eur. J. Bio­ [2]. The protein showed a potential activity to slow the chem., in press), both UAP and urinary bikunin were rate of CaOx crystal growth efficiently. Moreover, as isolated and purified from human urine. Their CaOx described in detail in reference [2], its crystallization growth inhibitory activity was assayed before and after inhibitory activity was greater than that of nephrocalcin pronase treatment. The inhibition assay consists in using or nephrocalcin-like macromolecule considered, at the a mixture of 1 ml supersaturated solution of CaCl2 (2 time, the principal urinary inhibitor of CaOx crystalliza­ mmol/1 in Tris 0.05 mol/l, NaCl 0.15 mol/1, pH 7 .3) in 45 tion [42]. The inhibitor was named uronic-acid-rich pro­ the presence of [ Ca]CaC12 and 1 ml ammonium oxa­ tein (UAP) due to its uronic acid contents. The molecu­ late (2 mmol/l in Tris 0.05 mol/1, NaCl 0.15 mol/l, pH lar characteristics of UAP indicated structural similari­ 7. 3). The tubes containing proteins were compared with ties with bikunin. The sequence of the first 18 amino those without added proteins. At the end of the assay, acid residues of the protein was identical with that of tubes were centrifuged at 2000 g for 5 minutes and 0.5 bikunin [l, 4]. Moreover, by using a polyclonal fol an­ ml of supernatant was withdrawn for radioactivity deter­ tibody by immunoblotting analysis, a positive immuno­ mination in a liquid scintillation counter. The inhibitory reaction was observed confirming the close relationship activity was determined before and after enzymatic di­ between UAP and fol [l]. gestion. The results shown in Figure 2 demonstrate that UAP was also isolated from rat urine and compared UAP exhibited a strong CaOx growth inhibitory activity to that isolated from human urine [l]. Both showed while urinary bikunin was less inhibitory. The CaOx identical amino acid composition, similar amino acid se­ inhibitory activity of both proteins disappeared after quence, and similar crystallization inhibitory activity. pronase digestion. Structurally, UAP and urinary bi­ UAP was assayed on CaOx crystal growth model before kunin have many similarities. Their molecular weight

428 Inter-a-inhibitor in urolithiasis

80 a human urinary protein which we named nephrocalcin­ like [2] due to its biochemical similarities with nephro­ a calcin described by Nakagawa et al. [42]. The molecu­ lar weight of the protein was about 16 kDa, it was able 60 to bind to Tamm-Horsfall protein, and it showed an im­ ,::: portant inhibitory activity toward CaOx crystallization. ._g However, this inhibitory activity was lower than that of -~,g UAP. Unfortunately, at that time, we did not determine :.a 40 its amino acid sequence. In a recent study, Ryalls ...... group [66] reported the partial amino acid sequence of ~ b a protein claimed to be nephrocalcin. It showed an identity with HI-14, a fragment ofbikunin. As we men­ 20 tioned earlier, prolonged digestion of fol or bikunin with trypsin, results in the production of two peptides, Hl-14

C and HI-8. Consequently, HI-14 is not a physiological d product. Moreover, many features of nephrocalcin do not match with Hl-14 fragment. For instance, nephro­ calcin has 2-3 ')'-carboxyglutamic acid (Gla) residues and Concentration (µg/ml) is synthesized in kidney, but Hl-14 does not contain Gla and is not produced in kidney. Therefore, the results Figure 2. Calcium oxalate growth inhibitory activity of obtained by Ryalls group remain speculative. UAP and urinary bilcunin before (a, c) and after treat­ ment (b, d) with pronase respectively. fol and its Related Proteins in Lithiasis Disease is 35 and 45 kDa respectively. They have similar amino Many reports indicate that urine of stone formers acid composition and similar amino acid sequence of contains macromolecules that inhibit CaOx crystal for­ first 18 residues. Furthermore, they are immunological­ mation, but not efficiently as the same inhibitors found ly related to fol. However, functionally they appear in the urine of normals [3 , 27 , 43]. With regards to different. A comparison of physico-chemical proprieties fol, its plasma concentration was reduced during various of UAP and urinary bilcunin is reported in Table 2. pathologic conditions including renal failure [24, 45 , 46, As we have reported above, bikunin is present in 68]. Simultaneously, urinary bilcunin concentration was free form or attached to different complexes related to increased suggesting an enhancement of fol turnover. fol. The free form can easily pass into urine through Although, these findings were not confirmed in other glomeruli due to its low molecular weight. Metabolism studies suggesting that fol and its related proteins could oflal or its related proteins (enzymatically for instance) be synthesized in separate pathways [13, 47, 58]. Ac­ may yield another form of bikunin. The latter may be cordingly, since UAP is related to fol, may be its excre­ modified during this process and present some differ­ tion is increased in the urine of patients with renal dis­ ences compared to the preexisting free form of bikunin. ease. Thereby, an increase in Ca Ox growth inhibitory Furthermore, the isolation of bikunin with different mo­ activity is anticipated. Unfortunately, this was not the lecular weights, has been reported by several investiga­ case, suggesting that UAP obtained from stone formers tors [7, 9, 16, 23, 61]. These discrepancies may be due may be structurally abnormal and does not inhibit crystal to differences in various technical procedures used in the precipitation efficiently. Accordingly, we have isolated isolation and purification of proteins. The carbohydrate UAP from the urine of stone formers and its inhibitory content of the protein and the presence of a GAG chain activity was compared to that purified from the urine of that can modify its electrophoretic mobility should be healthy subjects [3]. The results demonstrated that UAP taken into consideration since many results are still in obtained from stone patients exhibited less inhibitory ac­ disagreement. However, it has been reported that the tivity. Partial structure of this glycoprotein showed that carbohydrate chain does not contribute to CaOx inhibi­ it contained less sialic acid than that of normals [3]. tory activity of proteins related to fol [2, 63]. Experi­ ments are in progress to clarify this matter. fol Related Proteins in Stones Nephrocalcin with a molecular weight of about 14 kDa, is one of the well-known urinary inhibitors of Regardless of mineral composition, all urinary CaOx crystallization [42]. So far, the amino acid se­ stones contain an organic material refereed to as matrix quence of this inhibitor remains unknown. We isolated protein [35, 36]. The latter represents about 2-5 % by

429 F. Atmani, J. Miron and S.R. Khan weight and consists predominantly of proteins [12]. Its [5] Atmani F, Opalko FJ, Khan SR (1996) Associa­ role in the formation of urinary stones remains a subject tion of macromolecule with calcium oxalate crystals in­ of research. To better understand this role, many inves­ duced in vitro in normal human and rat urine. Urol Res tigators attempted to analyze proteins extracted from uri­ 24: 45-50. nary stones. Unfortunately, few works were devoted to [6] Aubry M, Bieth J (1976) A kinetic study of the a search for fol and its related peptides and to quantify inhibition of human and bovine trypsins and chymotryp­ them. Petersen et al. [ 48] have found hemoglobin and sins by the inter-a-trypsin inhibitor from human plasma. two serine proteases in the extract of CaOx kidney Biochim Biophys Acta 438: 221-230. stone. A protease inhibitor, al-antitrypsin, has been [7] Balduyck M, Hayem A, Kerckaert JP, Miron C, extracted and quantified suggesting that some stones are Miron J (1982) Isolation of human urinary trypsin in­ in contact with blood during their development [70]. hibitor. Biochem Biophys Res Comm 109: 1247-1255. Hoyer [31] has extracted several proteins from CaOx [8] Balduyck M, Laroui S, Miron C, Miron J monohydrate stones and quantified them. His results (1989) A proteoglycan related to the urinary trypsin in­ showed that uropontin, or osteopontin, constituted the hibitor (UTI) links the two heavy chains of inter-a-tryp­ major protein in the extract (90 µg/100 mg). Tamm­ sin inhibitor. Biol Chem Hoppe-Seyler 370: 331-336. Horsfall protein, albumin, and fol fragment represent [9] Balduyck M, Miron C, Loutfi H, Richet C, less than 10 µg/100 mg of stones. Recently, in our lab­ Roussel P, Miron J (1986) The major human urinary oratory we identified proteins extracted from CaOx crys­ trypsin inhibitor is a proteoglycan. Eur J Biochem 158: tals produced in vitro in human urine by adding sodium 417-422. oxalate [5]. Among the proteins, we detected a frag­ [10] Bourguignon J, Sesboiie R, Diarra-Mehrpour ment of fol, which could be bikunin, but in lesser M, Daveau M, Martin JP (1989) Human inter-a-trypsin amounts compared to prothrombin Fl and osteopontin. inhibitor. Synthesis and maturation in hepatoma HepG2 More experiments are in progress. cells. Biochem J 261: 305-308. [11] Bourguignon J, Vercaigne D, Sesboiie R, Concluding Remarks Martin JP, Salier JP (1983) Inter-alpha-trypsin inhibitor (ITI): Two mRNAs in baboon liver argue for a discrete It seems that UAP is biochemically similar to biku­ synthesis of ITI and ITI derivatives. FEBS Letters 162: nin, the light chain of fol. But functionally these two 379-383. proteins appear different. fol becomes a family of pro­ [12] Boyce WH (1968) Organic matrix of human teins involved in modulating calcium oxalate crystalliza­ urinary concretions. Am J Med 45: 673-683. tion and stone formation. Another plasma protein, the [13] Bratt T , Olsson H, Sjoberg EM, Jergil B, F 1 fragment of prothrombin has also been implicated in Akerstrom B (1993) Cleavage of a 1-microglobulin-bi­ calcium oxalate stone formation [64]. Apparently, the kunin precursor is localized to the Golgi apparatus of rat crystallization of calcium oxalate is modulated by a vari­ liver cells. Biochim Biophys Acta 1157: 147-154. ety of macromolecules including many plasma proteins. [14] Brissenden JE, Cox DW (1982) az-macroglob­ ulin production by cultured human fibroblasts. Som Cell References Gen 8: 289-305. [15] Businaro R, Leali FMT, DeRenzis G, Pompili [1] Atmani F, Khan SR (1995) Characterization of E, Pagliari G, Menghi G, Fumagalli L (1992) Inter-al­ uronic-acid-rich inhibitor of calcium oxalate crystalliza­ pha-trypsin inhibitor-related immunoreactivity in human tion isolated from rat urine. Urol Res 23: 95-101. tissues and body fluids. Cell Mol Biol 38: 436-471. [2] Atmani F, Lacour B, Driieke T, Daudon M [16] Chawla RK, Lawson DH, Ahmad M, Travis J (1993) Isolation and purification of a new glycoprotein (1992) Cancer-related urinary proteinase inhibitor, from human urine inhibiting calcium oxalate crystalliza­ EDCl: A new method for its isolation and evidence for tion. Urol Res 21: 61-66. multiple forms. J Cell Biochem 50: 227-236. [3] Atmani F, Lacour B, Jungers P, Driieke T, [17] Chiral F, Balduyck M, Miron C, Laroui S, Daudon M (1994) Reduced inhibitory activity ofuronic­ Sautiere P, Miron J (1991) A chondroitin-sulfate chain acid-rich protein in urine of stone formers. Urol Res 22: is located on serine-10 of the urinary trypsin inhibitor. 257-260. Int J Biochem 23: 1201-1203. [4] Atmani F, Lacour B, Strecker G, Parvy P, [18] Daveau M, Rouet P, Scotte M, Hiron M, Driieke T, Daudon M (1993) Molecular characteristics Lebreton JP, Sallier JP (1993) Human inter-a-trypsin of uronic-acid-rich protein, a strong inhibitor of calcium inhibitor family in inflammation: Simultaneous synthesis oxalate crystallization in vitro. Biochem Biophys Res of positive and negative acute-phase proteins. Biochem Comm 191: 1158-1165. J 292: 485-492.

430 Inter-a-inhibitor in urolithiasis

[19] Diarra-Mehrpour M, Bourguignon J, Sesboiie inter-a-trypsin inhibitor. I. Determination of the amino R, Mattei MG, Passage E, Salier JP, Martin JP (1989) acid sequence of the antitryptic domain by solid-phase Human plasma inter-a-trypsin inhibitor is encoded by Edman degradation. Hoppe-Seyler's Z Physiol Chem four genes on three chromosomes. Eur J Biochem 179: 360: 1285-1296. 147-154. [31] Hoyer JR (1994) Uropontin in urinary calcium [20] Dietl T, Dobrinski W, Hochstrasser K (1979) stone formation. Miner Electrolyte Metab 20: 385-392. Human inter-a-trypsin inhibitor. Limited proteolysis by [32] Jessen TE, Faarvang KL, Ploug M (1988) Car­ trypsin, plasmin, kallikrein and granulocytic elastase and bohydrate as covalent crosslink in human inter-a-trypsin inhibitory properties of the cleavage products. Hoppe­ inhibitor: A novel plasma protein structure. FEBS Let­ Seyler' s Z Physiol Chem 360: 1313-1318. ters 230: 195-200. [21] Enghild JJ, Salvesen G, Th0gersen IB, [33] Kastem W, Bjorck L, Akerstrom B (1986) De­ Valnickova Z, Pizzo SV, Hefta SA (1993) Presence of velopmental and tissue-specific expression of a 1-micro­ the protein-glycosaminoglycan-protein covalent cross­ globulin mRNA in the rat. J Biol Chem 261: 15070- link in the inter-a-trypsin inhibitor-related proteinase 15074. inhibitor heavy chain 2/Bikunin. J Biol Chem 268: 8711- [34] Kaumeyer JF, Polazzi JO, Kotick MP (1986) 8716. The rnRNA for a proteinase inhibitor related to the HI- [22] Enghild JJ, Th0gersen IB, Pizzo SV, Salvesen 30 domain of inter-a-trypsin inhibitor also encodes a-1- G (1989) Analysis of inter-a-trypsin inhibitor and a microglobulin (protein HC). Nucl Acids Res 14: 7839- novel trypsin inhibitor, Pre-a-trypsin inhibitor, from 7850. human plasma. Polypeptide chain stoichiometry and as­ [35] Khan SR, Hackett RL (1987) Crystal-matrix sembly by glycan. J Biol Chem 264: 15975-15981. relationships in experimentally induced urinary calcium [23] Fex G, Grubb A, Loeffler C, Larsson J (1981) oxalate monohydrate crystals, an ultrastructural study. Isolation and partial characteri:zation of a low molecular Calcif Tissue Int 41: 157-163. weight trypsin inhibitor from human urine. Biochim [36] Khan SR, Hackett RL (1993) Role of organic Biophys Acta 667: 303-308. matrix in urinary stone formation: an ultrastructural [24] Gebhard W, Hochstrasser K (1986) Inter-a­ study of crystal matrix interface of calcium oxalate trypsin inhibitor and its close relatives. In: Proteinase monohydrate stones. J Urol 150: 239-245. Inhibitors. Elsevier, Amsterdam. pp. 389-401. [37] Lindqvist A, Bratt T, Altieri M, Kastem W, [25] Gebhard W, Schreitmiiller T, Hochstrasser K, Akerstrom B (1992) Rat a 1-microglobulin: Co­ Wachter E (1989) Two out of the three kinds of subunits expression in liver with the light chain of inter-a-trypsin of inter-a-trypsin inhibitor are structurally related. Eur inhibitor. Biochim Biophys Acta 1130: 63-67. J Biochem 181: 571-576. [38] Malki N, Balduyck M, Maes P, Capon C, [26] Gressier B, Balduyck M, Mizon C, Mizon J Mizon C, Han KK, Tartar A, Fournet B, Mizon J (1990) Crossed immunoelectrophoresis does not allow (1992) The heavy chains of human plasma inter-a-tryp­ accurate determination of inter-a-trypsin inhibitor and its sin inhibitor: Their isolation, their identification by derivatives in plasma. Biol Chem Hoppe-Seyler 371: electrophoresis and partial sequencing. Biol Chem 865-870. Hoppe-Seyler 373: 1009-1018. [27] Hess B, Nakagawa Y, Parks JH, Coe FL [39] McKeehan WL, Sakagami Y, Hoshi H, (1991) Molecular abnormality of Tamm-Horsfall glyco­ McKeehan KA (1986) Two apparent human endothelial protein in calcium oxalate nephrolithiasis. Am J Physiol cell growth factors from human hepatoma cells are tu­ 260: F569-F578. mor-associated proteinase inhibitors. J Biol Chem 261: [28] Hochstrasser K, Bretzel G, Feuth H, Hilla W, 5378-5383. Lempart K (1976) The inter-a-trypsin inhibitor as pre­ [40] Morelle W, Capon C, Balduyck M, Sautiere P, cursor of the acid-stable proteinase inhibitors in human Kauach M, Michalski C, Fournet B, Mizon J (1994) serum and urine. Hoppe-Seyler's Z Physiol Chem 357: Chondroitin sulfate covalently cross-links the three poly­ 153-162. peptide chains of inter-a-trypsin inhibitor. Eur J Bio­ [29] Hochstrasser K, Schonberger OL, Rossmanith chem 221: 881-888. I, Wachter E (1981) Kunitz-type proteinase inhibitor de­ [41] Morii M, Travis J (1985) The reactive site of rived by limited proteolysis of the inter-a-trypsin inhibi­ human inter-a-trypsin inhibitor is in the amino-terminal tor. V. Attachments of carbohydrates in the human uri­ half of the protein. Biol Chem Hoppe-Seyler 366: 19-21. nary trypsin inhibitor isolated by affinity chromatogra­ [42] Nakagawa Y, Abram V, Kezdy FJ, Kaiser ET, phy. Hoppe-Seyler's Z Physiol Chem 362: 1357-1362. Coe FL (1983) Purification and characteri:zation of the [30] Hochstrasser K, Wachter E (1979) Kunitz-type principal inhibitor of calcium oxalate monohydrate crys­ proteinase inhibitor derived by limited proteolysis of the tal growth in human urine. J Biol Chem 258: 12594-

431 F. Atmani, J. Miron and S.R. Khan

12600. scription of the four liver-specific genes for inter-a-tryp­ [43] Nakagawa Y, Abram V, Parks JH, Lau HSH, sin inhibitor family in mouse. Biochem J 296: 85-91. Kawooya JK, Coe FL (1985) Urine glycoprotein crystal [56] Salier JP, Diarra-Mehrpour M, Sesboiie R, growth inhibitors. Evidence for a molecular abnormality Bourguignon J, Benarous R, Ohkubo I, Kurachi S, in calcium oxalate nephrolithiasis. J Clio Invest 76: Kurachi K, Martin JP (1987) Isolation and characteriza­ 1455-1462. tion of cDNAs encoding the heavy chain of human inter­ [44] 0dum L (1989) Immunohistochemical investi­ a-trypsin inhibitor (IaTI): Unambiguous evidence for gation of inter-a-trypsin inhibitor in urinary tract. multipolypeptidechain structure oflaTI. Proc Natl Acad APMIS 97: 375-360. Sci USA 84: 8272-8276. [45] 0dum L (1990) Inter-a-trypsin inhibitor and [57] Salier JP, Martin JP, Lambin P, McPhee H, pre-a-trypsin inhibitor in health and disease. Determi­ Hochstrasser K (1980) Purification of human serum in­ nation by immunoelectrophoresis and immunoblotting. ter-a-trypsin inhibitor by zinc chelate and hydrophobic Biol Chem Hoppe-Seyler 371: 1153-1158. interaction chromatographies. Anal Biochem 109: 273- [46] 0dum L (1991) Investigation of inter-a-trypsin 283. inhibitor and slow migrating proteinase inhibitors in [58] Salier JP, Sesboiie R, Vercaigne D, serum and urine. Danish Med Bull 38: 68-77. Bourguignon J, Martin JP (1983) Inter-a-trypsin inhibi­ [47] 0dum L, Hansen-Nord G, Byrjalsen I (1987) tor (ITI): Use of a new antisera for quantitative studies Human inter-a-tr;psin inhibitor and immunologically re­ and discrete quantitation of ITI and its derivatives. lated inhibitors investigated by quantitative immunoelec­ Annal Biochem 133: 336-343. trophoresis. IL Pathological conditions. Clio Chim Acta [59] Salier JP, Simon D, Rouet P, Raguenez G, 162: 189-198. Muscatelli F, Gebhard W, Guenet JL, Mattei MG [48] Petersen TE, Th0gersen I, Petersen SE (1989) (1992) Homologous chromosomal locations of the four Identification of hemoglobin and two serine proteases in genes for inter-a-trypsin inhibitor and pre-a-inhibitor acid extracts of calcium containing kidney stones. J Urol family in human and mouse: Assignment of the ancestral 142: 176-180. gene for the lipocalin superfamily. Genomics 14: 83-88. [49] Pierzchalski P, Rokita H, Koj A, Fries E [60] Schreitmiiller T, Hochstrasser K, Reisinger (1992) Synthesis of a 1-microglobulin in cultured hepato­ PWM, Wachter E, Gebhard W (1987) cDNA cloning of cytes is stimulated by interleukin-6, leukemia inhibitor human inter-a-trypsin inhibitor discloses three different factor, dexamethasone and retinoic acid. FEBS Letters proteins. Biol Chem Hoppe-Seyler 368: 963-970. 298: 165-168. [61) Selloum L, Davril M, Miron C, Balduyck M, [50] Potempa J, Kwon K, Chawla R, Travis J Miron J (1987) The effect of the glycosaminoglycan (1989) Inter-a-trypsin inhibitor. Inhibition spectrum of chain removal on some properties of the human urinary native and derived forms. J Biol Chem 264: 15109- trypsin inhibitor. Biol Chem Hoppe-Seyler 368: 47-55. 15114. [62] Sjoberg EM, Fries E (1992) Biosynthesis of [51] Reisinger P, Hochstrasser K, Albrecht GJ, bikunin (urinary trypsin inhibitor) in rat hepatocytes. Lempart K, Salier JP (1985) Human Inter-a-trypsin in­ Arch Biochem Biophys 295: 217-222. hibitor: Localization of the Kunitz-type domains in the [63] S0rensen S, Hansen K, Bak S, Justesen SJ N -terminal part of the molecule and their release by a (1990) An unidentified macromolecular inhibitory con­ trypsin-like proteinase. Biol Chem Hoppe-Seyler 366: stituent of calcium oxalate crystal growth in human 479-483. urine. Urol Res 18: 373-379. [52] Rouet P, Daveau M, Salier JP (1992) Electro­ [64] Stapleton AMF, Ryall RM (1995) Blood coagu­ phoretic pattern of the inter-a-trypsin inhibitor family lation proteins and urolithiasis are linked: Crystal matrix proteins in human serum, characterized by chain-specific protein is the F 1 activation peptide of human pro­ antibodies. Biol Chem Hoppe-Seyler 373: 1019-1024. thrombin. Br J Urol 75: 712-719. [53] Rouet P, Raguenez G, Tronche F, Yaniv M, [65] Steinbuch M (1976) The inter-a-trypsin N'Guyen C, Salier JP (1992) A potent enhancer made of inhibitor. Methods Enzymol 45: 760-772. clustered liver-specific elements in the transcription [66] Tang Y, Grover PK, Moritz RL, Simpson RJ, control sequences of human a 1-microglobulin/bikunin Ryall RL (1995) Is nephrocalcin related to the urinary gene. J Biol Chem 267: 20765-20773. derivative (bikunin) of inter-a-trypsin inhibitor? Br J [54] Salier JP (1990) Inter-a-trypsin inhibitor: Urol 75: 425-430. Emergence of a family within the Kunitz-type protease [67] Tavakkol A (1991) Molecular cloning of por­ inhibitor superfamily. TIBS 15: 435-439. cine a 1-micoglobulin/Hl-30 reveals developmental and [55] Salier JP, Chan P, Raguenez G, Zwingman T, tissue-specific expression of two variant messenger ribo­ Erickson RP (1993) Developmentally regulated tran- nucleic acids. Biochim Biophys Acta 1088: 47-56.

432 Inter-a-inhibitor in urolithiasis

[68] Toki N, Sumi H (1982) Urinary trypsin inhibi­ dues. However, Hl-14 or any protein related to fol tor and urokinase activities in renal diseases. Acta does not have any Gia residue. Interestingly, in a recent Haemat 67: 109-113. work, Tang et al. [66) reported that NC is Hl-14 by us­ [69] Travis J, Salvesen GS (1983) Human plasma ing similar techniques described by Nakagawa to isolate proteinase inhibitors. Ann Rev Biochem 52: 655-709. NC. The authors suggested that NC was contaminated [70] Umekawa T, Kohri K, Amasaki N, Yamate T, by other proteins containing Gia, like prothrombin Fl, Yoshida K, Yamamoto K, Suzuki Y, Sinohara H, Kurita to explain the presence of these residues in the prepara­ T (1993) Sequencing of a urinary stone protein, identical tion of NC. Nonetheless, another feature of NC is it is to alpha-one antitrypsin, which lacks 22 amino acids. synthesized in kidney, but Hl-14 is not. Therefore, sim­ Biochem Biophys Res Comm 193: 1049-1053. ilarity or identity of NC to Hl-14 remains speculative [71] Vetr H, Gebhard W (1990) structure of the and needs confirmation by, e.g., using NC antibody. human a 1-microglobulin-bikunin gene. Biol Chem Hoppe-Seyler 371: 1185-1196. B. Hess: It should be acknowledged that there are cer­ [72) Wachter E, Hochstrasser K (1981) Kunitz-type tain requirements for an inhibitor/modifier to be of clini­ proteinase inhibitor derived by limited proteolysis of the cal significance for calcium stone formation, such as: (1) inter-a-trypsin inhibitor. IV. The amino acid sequence abundance in urine, (2) occurrence of calcium binding of the human urinary trypsin inhibitor isolated by affini­ sites in the molecule, (3) structural and functional abnor­ ty chromatography. Hoppe-Seyler's Z Physiol Chem malities or significantly reduced excretion in active stone 362: 1351-1355. formers versus healthy people, and (4) possibly some [73) Yoshida E, Sumi H, Maruyama M, Tsushima therapeutic implications, i.e., altering physico-chemical H, Matsuoka Y, Sugiki M, Mihara H (1989) Distribu­ conditions of urines, modifying the diet or prescribing tion of acid stable trypsin inhibitor immunoreactivity in certain drugs should improve the functional status of a normal and malignant human tissues. Cancer 64: 860- given macromolecule towards more inhibition of crystal­ 869. lization, mainly crystal aggregation. [74] Yoshida E, Sumi H, Tsushima H, Maruyama Authors: In this paper, we do not attempt to describe M, Mihara H (1991) Distribution and localization of in­ UAP as a superinhibitor to be considered and the others ter-a-trypsin inhibitor and its active component acids­ to be discarded. In the paper where UAP was described stable proteinase inhibitor: Comparative immuno­ for the first time [2], we pointed out that one inhibitor histochemical study. Inflammation 15: 71-79. alone cannot be responsible for the total urinary inhibi­ tory activity. Moreover, in a second paper [3], we re­ Discussion with Reviewers ported that UAP extracted from the urine of stone form­ ing subjects was less inhibitory compared to that isolated B. Hess: How do the authors explain the suggested re­ from the urine of healthy persons. We suggested that absorption of fol in the proximal tubule, i.e., how does this diminution does not account for the total urinary a protein with a molecular weight of 180-220 kDa get inhibitory activity, but that other protein inhibitors also across the tubular membrane into the tubular fluid? may be defective. Reviewer VII: The question of whether bikunin/UAP (or fol) is synthesized in the kidney, or is merely fil­ M. Daudon: The authors suggest that variation in uri­ tered at the glomerulus is not directly addressed. Do the nary excretion of fol related proteins may occur in vari­ authors have any data to define more precisely the ous pathological conditions responsible for an increased nature of the fol reactivity? excretion of bikunin. Do the authors have some infor­ Authors: fol is mainly synthesized in the liver and se­ mation about the normal range of UAP and bikunin ex­ creted in plasma. Recent work studying the expression cretion in human urine? of mRNA coding for fol confirmed its exclusive synthe­ Authors: The normal range of urinary excretion of fol sis in the liver [55]. The presence of a positive fol related proteins is about 2 to 10 mg/day according to the immunoreactivity in kidney is due to the presence of bi­ method used [77, 78). This excretion can be enhanced kunin which has a molecular weight in a range of 35-45 to 50-100 fold or even more in certain pathological kDa, and can easily pass through the glomerular filters, conditions such as cancer [75, 76). thus, it can be recognized by fol polyclonal antibody. M. Daudon: How can the authors explain the weaker B. Hess: Similarity of nephrocalcin (NC) and Hl-14 of inhibitory activity of UAP reported in this paper as fol/bikunin: nephrocalcin contains Gia; what is the Gia­ compared to that reported in a previous paper [4]? content of Hl-14 of fol/bikunin? Authors: The weaker inhibitory activity of UAP in this Authors: Nephrocalcin is known to have 2-3 Gia resi- study is not significant compared to that reported in the

433