Biosynthesis, processing, and sorting of human myeloperoxidase

Archives of Biochemistry and Biophysics 445 (2006) 214–224 www.elsevier.com/locate/yabbi

Review

Biosynthesis, processing, and sorting of human myeloperoxidase Markus Hansson a,¤, Inge Olsson a, William M. Nauseef b a

b

Department of Hematology, C14, BMC, SE-221 84 Lund, Sweden InXammation Program and Department of Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, IA 52242, USA Received 27 May 2005, and in revised form 8 July 2005 Available online 31 August 2005

Abstract Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating eYcient microbial killing but also can contribute to progressive tissue damage in chronic inXammatory states such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to Wnal proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulWde bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is unknown, the pro-segment is required for normal processing and targeting, as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally occurring mutations that cause inherited MPO deWciency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting.  2005 Elsevier Inc. All rights reserved. Keywords: Neutrophil; Monocyte; Intracellular sorting; Secretion; InXammation; Innate immunity; Lysosome; Calnexin; Calreticulin; Heme

Sixty-Wve years ago Agner isolated from purulent Xuid of patients with tuberculous empyema an iron-containing protein with peroxidase activity and named the newly discovered protein verdoperoxidase because of its intense green color [1]. Subsequent studies deWned the tissue distribution to be limited to myeloid cells, thus prompting the renaming of the protein myeloperoxidase (MPO).1 Thirty years after Agner’s discovery, KlebanoV demonstrated that MPO contributed to oxygen-dependent killing by phagocytes [2,3], thereby stimulating a generation of scientists to dissect the speciWc role of this protein in innate immunity. As the result of numerous ele-

*

Corresponding author. Fax: +46 46 184493. E-mail address: [email protected] (M. Hansson). 1 Abbreviations used: MPO, myeloperoxidase; TPO, thyroid peroxidase; LPO, lactoperoxidase; EPO, eosinophil peroxidase; Endo H, endoglycosidase H; AluRRE, Alu receptor response element; PPAR, peroxisome proliferator-activated receptor; CRT, calreticulin; CLN, calnexin; TGN, trans-Golgi network; M-6-P, mannose-6-phosphate; sTNFR, soluble TNF
receptor; Pro, propeptide. 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.08.009

gant studies, the antimicrobial action of MPO has been attributed to its unique capacity to produce hypochlorous acid and other toxic agents [4] that create an environment within the phagolysosome of neutrophils that inhibits or kills ingested microbes [5]. MPO utilizes H2O2 to eVect posttranslational modiWcations of target molecules, following a paradigm utilized by all members of the animal peroxidase family, although the capacity to oxidize Cl¡ to Cl+ at physiologic pH is a property unique to MPO (see later). Concomitant with release of MPO into the phagosome, the NADPH-dependent oxidase of phagocytes is activated to generate the required H2O2 for MPO to mediate HOCl generation. Other members of the animal peroxidase protein family, namely thyroid peroxidase (TPO), lactoperoxidase (LPO), and eosinophil peroxidase (EPO) follow the same fundamental paradigm, each using a diVerent H2O2 source. MPO-mediated damage is not limited to intraphagosomal microbes, as HOCl and its derivatives can damage host tissues as well. Thus MPO has been implicated in the pathogenesis of diverse inXammatory diseases, including

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atherosclerosis, demyelinating disorders of the central nervous system, and certain tumors [6]. In support of the occurrence of host toxicity, there is direct evidence of MPO-catalyzed modiWcations including tyrosyl radical formation [7–9], chlorination [10], tyrosine peroxide generation [11], and oxidation of serum lipoproteins [12–14]. For example, peroxidatively active MPO has been detected in arteriosclerotic lesions [15] and MPO-catalyzed lipoprotein oxidation has been implicated in the evolution of cardiovascular disease [16], consistent with the contribution of lipid-laden foam cell macrophages to subintimal damage typical of atherosclerosis [17]. Some epidemiologic studies suggest that individuals with inherited MPO deWciency have less cardiovascular disease than does the normal population, consistent with MPO-dependent biochemical events contributing to the initiation or progression of atherosclerosis [18]. In light of the roles of MPO in such a broad array of inXammatory diseases, research on the structure and function of members of the peroxidase family promises to provide fundamental understanding of pathophysiology of a variety of diseases with potential implications for treatment and prevention. In this review, we summarize current understanding of the biosynthesis, processing, and sorting of human peroxidases, extending information presented in previous reviews on this topic [5,6,19–22]. MPO storage MPO Wgures prominently in the antimicrobial action of neutrophils, the dominant eVector cell of the innate immune system and the prominent cell in acute inXammation. As such, neutrophils are geared to respond nearly instantaneously to threats posed by invading microbes or other challenges to the integrity of the host. To kill and degrade organisms, neutrophils ingest microbes into phagosomes where antimicrobial agents can be recruited. Under optimal conditions, the stimulated neutrophil coordinates several distinct cellular events to deliver bioactive molecules to the phagosome to the detriment of the ingested microbe. Coincident with phagocytosis is activation of the NADPH oxidase, a source of H2O2 and other reactive oxygen species [23–25], and release of granule contents. Thus the neutrophil is uniquely organized to respond quickly, generating reactive oxygen species and delivering stored granule proteins into the phagolysosome or the extracellular space upon stimulation [20,26]. As suggested by their classiWcation as granulocytes, neutrophils possess a large number of intracellular, membrane-bound granules. During their development in the bone marrow, myeloid precursors synthesize at least three distinct populations of storage granules; those that stain for peroxidase activity, called primary or azurophil granules, and two types of peroxidase-negative granules. The lysosome-like azurophil granules are manufactured by the promyelocyte and house several antimicrobial proteins, serine proteases, and lysosome hydrolases [20,26]. Most important

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for our discussion is the presence of MPO in these granules, where it represents its major constituent [26]. Hematopoietic cells commonly have lysosome-related organelles for storage of cytolytic proteins, such as MPO together with lysosome hydrolases [27]. Therefore, these organelles are also named secretory lysosomes to stress the combined ability for storage, regulated secretion and lysosomal activity [28]. Secretory lysosomes have a characteristic morphology with an external limiting membrane, internal membranebound vesicles, dense cores of tightly packed proteins, and a matrix compartment [28]. The internal vesicles may serve a lytic function, whereas the dense core and matrix compartments primarily may serve storage functions [28]. In addition to the azurophil granules, neutrophils possess secondary and tertiary granules, which are formed during the myelocyte stage of maturation [20,26]. In contrast to primary granules, these granules lack MPO and their membranes are a signiWcant reservoir for functionally important membrane proteins, including receptors for complement components and formyl peptides [26]. These three granule subtypes diVer in their composition as well as the dynamics of their degranulation. Secondary granules are mobilized more readily than are the MPO-containing primary granules [20,26]. In fact secondary granule release may occur as part of neutrophil activation during tissue transmigration [26]. MPO is released predominantly into the phagolysosome during degranulation and to a limited extent extracellularly. Immunochemical analysis of the peroxidase activity detected in plasma [29] reveals the presence of both mature MPO and the precursor form, the latter perhaps arising from constitutive secretion of MPO precursor during its biosynthesis in the bone marrow (see below). Although the azurophil granules uniformly stain for peroxidase activity, MPO-containing primary granules are not a homogeneous population, a reXection of asynchronous packaging during myeloid maturation or a peculiar feature of granule biogenesis. Although the basis for this structural organization is unknown, one can distinguish at least three types based on size, electron density, and intravesicular MPO distribution. In promyelocytes are small electron dense azurophilic granules and nucleated azurophilic granules, both with uniform MPO distribution, as well as large, spherical azurophilic granules with MPO detected at the rim along the granule periphery [30]. For all three types of azurophilic granules, the heterogeneity of their contents suggests that the contents are not randomly packed, but rather are organized by a dynamic process that includes sequential formation and maturational change to achieve the Wnal structure. Azurophilic granules contain glycosaminoglycans that are anionic molecules and as such may provide a matrix to bind the cationic contents in a conformation or state that renders the enzymes inactive [31]. MPO expression MPO is encoded in a single gene, spanning 14 kb on the long arm of chromosome 17, whose expression is

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restricted to myeloid cells [32]. During granulocyte diVerentiation in the bone marrow, only promyelocytes and promyelomonocytes actively synthesize MPO [20,26]. Accordingly, a variety of transcription factors regulate normal MPO gene expression in a tissue- and diVerentiation-speciWc manner and MPO synthesis terminates as myeloid progenitors enter the myelocyte stage of diVerentiation. The same coordination is seen in vitro when cultured human myeloid precursor cell lines, such as HL-60 or PLB-985 cells, are chemically induced to diVerentiate towards a granulocytic lineage. DiVerentiating agents, such as phorbol esters or trans-retinoic acid, result in cessation of MPO gene transcription and abrupt termination of MPO biosynthesis within hours. Although there are several reports of PCR ampliWcation of MPO transcripts from mature neutrophils [33], tissue monocytes, or macrophages [34], there is no evidence published demonstrating MPO protein synthesis by non-malignant cells, other than myeloid precursors in bone marrow. Nonetheless, these observations provide ample incentive for additional studies to identify combinations of cytokines or in vivo conditions that can initiate transcription of the MPO gene in these cells. SigniWcant advances have been made in understanding the molecular regulation of both normal and abnormal MPO production. Progressive demethylation in the 5⬘ Xanking region of the MPO gene provides the open chromatin structure required for transcription [35,36]. Alternative splicing of mRNA gives rise to transcripts of 3.6 and 2.9 kB [37]. The MPO gene is regulated by the transcription factor AML1, and consequently the integrity of an AML1 binding site is essential for the activity of the proximal enhancer [38,39]. The Reynolds’ laboratory has identiWed an allelic polymorphism, ¡463G/A, in the promoter region of the MPO gene [40]. This site contains an Alu receptor response element (AluRRE), which is recognized by various nuclear receptors including Sp1 [31,40]. When the Sp1 site is intact (i.e., ¡463G), there is a 25-fold greater rate of transcription of MPO than from –463A. Accordingly, the data predict that individuals with the ¡463GG genotype would have the greatest intracellular amount of MPO, whereas those with ¡463AA the least and heterozygous ¡463GA individuals with intermediate levels. Many epidemiologic studies have pursued to link the relative risk for a given inXammatory disease with the MPO promoter genotype [41–64]. However the results have varied among the populations examined and reveal a very strong inXuence of gender on the degree of association of MPO genotype with several of the inXammatory diseases studied. Thus it seems that MPO genotype may be but one important variable in inXuencing an individual’s risk of acquiring speciWc diseases and that the dissection of the interacting determinants will require additional studies of well-characterized populations. Recent evidence indicates that agonists for the peroxisome proliferator-activated receptor (PPAR) regulate MPO gene expression via the aforementioned AluRRE [31,40,65].

Pertinent to the putative contribution of MPO to inXammation, AluRRE has been implicated in the incidence of a variety of inXammatory disorders, including atherosclerosis, Alzheimer’s disease, and certain tumours and it is interesting that there are frequently very dramatic gender-speciWc associations in epidemiologic studies linking such inXammatory diseases with speciWc genotypes in the MPO promoter region. The PPAR and estrogen receptor compete for binding the AluRRE in the MPO promoter, perhaps providing an instructive molecular explanation for this provocative clinical observation. It is important to note as well that the murine MPO gene lacks the AluRRE speciWc for primates. This species diVerence in the MPO promoter may explain the observed failure of the MPO knock-out mouse to recapitulate the participation of MPO in atheromatous lesions, a feature of human disease [66,67], and provide a strong incentive to develop human MPO transgenic murine models to better mirror the human disease. In addition to promyelocytes, monocyte precursors also synthesize MPO during their maturation in the bone marrow. However, normal expression is limited to this stage of myeloid development. Circulating monocytes do not actively synthesize MPO and the diVerentiation of monocytes into macrophages seen in tissue is likewise accompanied by downregulation of MPO synthesis. However MPO gene expression can be reinitiated in these cells when in the proper tissue setting. For example, -amyloid, a component of Alzheimer’s plaques, induces MPO gene transcription in quiescent brain macrophages [50]. A large body of evidence implicates MPO in the pathogenesis of atherosclerotic disease, with clear immunological identiWcation of MPO at the diseased sites and with Wrm analytical chemistry demonstrating the presence of chlorotyrosine residues, biochemical “footprints” of MPO action, in the atherosclerotic plaque [14,68]. However, the origin of the MPO mediating these events has been unresolved, as neutrophils are not a prominent cell type in the plaque and normal macrophages express very little MPO. It is possible that the conditions within the nascent plaque are such that de novo MPO synthesis occurs in the local macrophages. PPAR is abundant in foam cell macrophages of atherosclerotic lesions [69], thereby providing a potential mechanism for MPO gene transcription [34]. Based on our limited understanding of normal MPO biosynthesis and intracellular sorting (vide infra), we would predict that the foam cells newly engaged in MPO gene transcription stimulated by local cytokine would lack the cellular machinery to proteolytically process proMPO into the subunits of mature MPO, resulting in all of the proMPO entering the secretory pathway and exiting the cell. Thus, enzymatically active proMPO would be present in the atheroma and thus available to catalyze the posttranslational modiWcation of proteins and lipoproteins to promote atherosclerosis. Further study is needed to test this prediction, identify the cellular origins of the MPO, and deWne precisely to what extent MPO contributes to atherosclerotic disease.

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enzymes, MPO precursors undergo a complex series of proteolytic processing events during their synthesis and intracellular traYcking to achieve the Wnal structure of the native protein [70–77] (Fig. 1). As is typical for all N-linked oligosaccharides [78], the carbohydrate side chains in MPO are added en bloc to the nascent protein in the ER as a unit,

MPO biosynthesis: events in the endoplasmic reticulum Analysis of the sequence of the cDNA for MPO predicts many features typical of glycoproteins, including a putative signal peptide and several consensus sequences for asparagine-linked glycosylation [32]. Like many glycoprotein

A

B

pro

preproMPO

ER apoproMPO

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pro

pro

pro

Signal peptide

CRT apoproMPO

pro

TGN CLN proMPO

pro

-S-S-

-S-S-

pro

-S-S-

proMPO

Primary granules -S-S-

-S-S-

-S-S-

mature MPO

pro

Constitutive secretion

C

ER degradation high mannose oligosaccharide side chain complex oligosaccharide side chain

TGN

heme group

Primary granules

Constitutive secretion Fig. 1. MPO biosynthesis, processing and traYcking. (A) MPO is synthesized as a single primary 80 kDa translation product containing the sequences the propeptide (pro), the large subunit
and the small subunit . Cotranslational cleavage of the signal peptide and incorporation of high mannose oligosaccharide side chains yields the 90 kDa apoproMPO, which associates sequentially with the molecular chaperones CRT and CLN still in the ER. During association with calnexin, apoproMPO acquires heme and becomes enzymatically active proMPO. A series of proteolytic steps follow during which the propeptide is removed, a hexapeptide is excised between the heavy and light subunits, the terminal serine residue is deleted, and the resultant protein is cleaved into the two subunits coupled into followed by dimerization to produce mature MPO. (B) Heme-containing proMPO is formed in the ER. Some complex carbohydrate side chains are added in the TGN to constitutively secreted part of proMPO. The propeptide is removed in late endosomes or granules, and mature dimeric MPO with high mannose carbohydrates is formed in granules. (C) A propeptide-deleted mutant MPO is partially degraded in ER and partially exported followed by full accomplishment of complex oligosaccharide side chains in TGN. All product is constitutively secreted and no granule targeting occurs.

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each containing two N-acetylglucosamine residues, nine mannoses, and three glucoses (GlcNAc2Man9Glu3). In the ER there is limited deglucosylation with removal of terminal glucoses and one mannose group by the action of ER glucosidases and ER mannosidase, respectively, and the glycoprotein exiting the ER has no new sugars added. The resulting side chains are considered “high mannose oligosaccharides,” indicating the presence only of two N-acetylglucosamines and eight or fewer mannoses, and are susceptible to digestion with endoglycosidase H (endo H). The stepwise processing from precursor to mature MPO has been investigated by metabolic amino acid radiolabelling (radiolabel pulse) of immature granulopoietic cells, followed by prolonged incubation with excess unlabelled amino acid (radiolabel chase) in order to visualize the molecular changes coincident with MPO maturation over time. Changes in size secondary to processing are visualized by SDS–polyacrylamide gel electrophoresis. Data from several investigators [70–77] support predictions based on MPO cDNA clones [32,37,79–82]. Based on the comparison of cDNA for MPO and the amino acid sequence for the mature protein, there are three proteolytic events that accompany MPO maturation: an amino-terminal 125 amino acid propeptide is deleted, a small hexapeptide (ASKVTG) between the light and heavy chains is excised, and the carboxy-terminal serine residue is removed [37]. In addition, insertion of heme involves formation of three new covalent bonds and critically inXuences the structure of MPO intermediates during processing. The Wrst steps in MPO biosynthesis convert the 80 kDa primary translation product into 90 kDa apoproMPO after cotranslational cleavage of a signal peptide, N-linked glycosylation, and limited deglucosylation of high mannose oligosaccharide side chains. ApoproMPO lacks heme, is consequently without peroxidase activity, and appears to have a very long half-life in the ER (reviewed in [5,19,20]). There are Wve predicted sites for N-linked glycosylation in the amino acid sequence of apoproMPO, at asparagine residues 139, 323, 355, 391, and 483.2 The X-ray crystal structure indicates only three glycosylation sites, although biosynthetic studies demonstrate four glycosylated asparagine residues (WMN, unpublished results). With the exception of N139, which resides in the propeptide, all glycosylated residues are predicted to be in the heavy subunit, an arrangement conWrmed by the experimental data. Although the functional consequences of MPO glycosylation are not deWned, the oligosaccharides added to apoproMPO contribute to its transient associations with the ER molecular chaperones calreticulin (CRT), calnexin (CLN), and ERp57 [83]. Both CRT and CLN are high capacity, low aYnity calcium-binding proteins that reside in the ER [83]. During transit in the ER, nascent glycopro2 The amino acids are numbered from the initial methionine of the precursor form of MPO and thus include 786 amino acids. Accordingly, they include the 166 amino acids in the pre and pro regions that are not present in the crystal structure analyses of the mature protein.

teins, including MPO, undergo limited deglucosylation and modiWcations as part of a sequence of interactions with the molecular chaperones to promote proper folding and institute a level of quality control in glycoprotein synthesis. Although CLN is a transmembrane protein, its intraluminal domains share sequence homology and structural organization with CRT, a soluble protein in the ER lumen. Given the structural similarities and the Wrm evidence that CLN functioned as a molecular chaperone for glycoprotein synthesis, it was long suspected that CRT likewise served this function in the ER. The interaction of CRT with MPO was the Wrst demonstration that CRT was a molecular chaperone in the ER [84], a Wnding since conWrmed for a wide variety of proteins [83]. ApoproMPO interacts both with CRT and CLN during its prolonged residence in the ER and, as is true for other proteins, these interactions are not exclusively lectin-mediated. Metabolic inhibitors of ER glucosidases inhibit the trimming of terminal glucoses on apoproMPO but do not abrogate CRT-apoproMPO association, indicating that interactions in addition to those that are lectin-dependent participate in apoproMPO-CRT binding, as demonstrated for other glycoproteins in the ER [85]. To date it is not known to what extent, if any, the transient associations of CRT and CLN with apoproMPO are necessary for the subsequent events in MPO maturation. All of the events described thus far occur in the ER. When the Golgi is disrupted by treating cultured promyelocytes with brefeldin A [86], MPO biosynthesis is arrested at the stage of proMPO; heme is inserted normally but there is no proteolytic processing to mature MPO or secretion of precursor into the medium. Thus heme incorporation occurs in the ER, with subsequent exit into the Golgi and the distal secretory pathway. Furthermore, the sequential processing form of 90 kDa proMPO requires heme insertion to proceed towards full maturation, as inhibition of heme synthesis not only eliminates peroxidase activity but also prevents ER export, proteolytic processing, and granule targeting [86]. Therefore, heme is essential for peroxidase activity and for making proMPO properly folded and competent to exit the ER, able to reach late endosomes and granules for Wnal processing and storage. Heme incorporation in MPO For years the precise nature of the heme group and its ligation with the polypeptide eluded investigators and served as stimuli for extensive analysis of the biophysical and spectral properties of the puriWed protein. Data from the crystal structure and from a variety of analytical studies demonstrate that the prosthetic group in MPO is derived from FeIII protoporphyrin IX. All members of the animal peroxidase family share the unusual feature of having two or three covalent bonds between the prosthetic group and the protein backbone. All members have two ester linkages, one between the carboxyl group of a glutamate and a hydroxyl group on the 1-methyl substituent and the other between the carboxyl group of an aspartate and the

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hydroxyl group of the 5-methyl substituent. Unique to MPO is the presence of a third covalent bond, a sulfonium linkage between the sulfur of methionine 409 and the -carbon of the 2-vinyl substituent. There are two consequences of this additional covalent bond. Whereas the Soret band of the other family members is 412 nm, MPO is red-shifted to 430 nm. Presumably this unique conWguration in the heme pocket accounts for the unique capacity of MPO to chlorinate targets at physiologic pH, a reXection of its higher oxidation potential. These three covalent bonds in the animal peroxidases form through an autocatalytic process, although most of the mechanistic details remain unknown [87–90]. It is equally unclear what evolutionary advantages are gained by the covalent heme ligation, as the substrate speciWcity of the animal peroxidases is shared by unrelated peroxidases that lack covalently bound heme. MPO proteolytic maturation, intracellular traYcking and targeting Subsequently the enzymatically active proMPO undergoes the Wrst in a series of proteolytic events, being converted to a 74 kDa short-lived intermediate that lacks the 125 amino acid propeptide [72,74,91]. Either in or en route to the primary granule, the 74 kDa transient intermediate is cleaved into a two-subunit form comprised of a 59 kDa
subunit and a 13.5 kDa -subunit that are linked via the covalent bonds associated with the heme group, as discussed above. Two heavy-light protomer units then interact to produce the mature form of MPO, a symmetric homodimer of approximately 150 kDa with each half linked by a disulWde bond between C319 residues of the heavy subunit [92–94]. Mature dimeric MPO is stored in primary granules, but the functional consequences of dimer formation are unknown. It is noteworthy that MPO is the only member of the animal peroxidase family that is a dimer. After transport from the ER and through the complex trans-Golgi network (TGN), MPO precursors must reach their Wnal intracellular destination in the azurophilic granule or exit into the extracellular space (Fig. 1). To achieve these ends, the cell must segregate those proteins destined for the intracellular compartment from those to be secreted from the cell. As the latter pathway is constitutive, the cell must tag and retrieve proteins from the secretory pathway and redirect them to the target organelle. In most cells these events are achieved by one of two mechanisms; either directly by transport through the TGN to late endosomes and on to lysosomes, or indirectly by targeting Wrst to the plasma membrane with subsequent internalization into early endosomes and late endosomes [95]. In many cells hydrolases and granzymes use the mannose-6-phosphate (M-6-P) receptor system for targeting proteins to lysosomes [96,97] and protein aggregation facilitates secretory granule targeting in exocrine and endocrine cells [98,99]. However, neither the M-6-P receptor system [100] nor protein aggregation has been proven to be important for protein sorting in myeloid cells. In fact, ligand-controlled intracellular olig-

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omerization does not directly facilitate lysosome targeting [101]. We know that myeloid precursors constitutively secrete monomeric proMPO [102], although the majority of proMPO is retained intracellularly, processed and delivered to the azurophilic granule by mechanisms incompletely characterized. As mentioned earlier, glycoproteins exit the ER as high mannose units, still susceptible to digestion with Endo H. During their passage through the Golgi they are exposed to a variety of transferases and glucosidases that extensively modify the oligosaccharide units, including the deletion of mannoses, addition of galactose, fucose, or sialic acid residues, and posttranslational modiWcations such as phosphorylation and sulfation. The resulting structures are termed “complex oligosaccharides” to reXect their structural diversity and are resistant to Endo H digestion. Thus, susceptibility to Endo H digestion distinguishes between high-mannose and complex oligosaccharides. Although the conversion of apoproMPO to proMPO in the ER is extremely slow, processing in the Golgi, granule targeting, and secretion are rapid. As noted above, the formation of complex carbohydrate side chains is restricted to median and trans-Golgi, so the acquisition of complex oligosaccharides on a glycoprotein serves as evidence that it has traversed the TGN sorting station. In general, granule proproteins travel through Golgi from cis to trans for incorporation into transport vesicles sculpted out at TGN and bound for late endosomes and granules. However the oligosaccharides on mature MPO and proMPO diVer in their susceptibility to Endo H. Whereas the oligosaccharides on mature MPO are susceptible to Endo H, secreted proMPO is partially Endo H resistant [103]. These data suggest that MPO targeted to the azurophilic granule and proMPO destined to be secreted have diVerent conformations during transit through the Golgi, such that the former are not exposed to oligosaccharide-processing enzymes whereas the latter are. The basis for these diVerent fates and the structural implications for the oligosaccharide diVerence between intracellular and extracellular forms are unknown. Several proteins localized to azurophilic granules are proteolytically modiWed to their mature forms after exit from the TGN. During sorting of MPO, the MPO propeptide is removed before or during Wnal processing in azurophil granules. Thus, it is possible that pro-segments contribute to the correct transport through the biosynthetic machinery, but not necessarily for the speciWc sorting process. As mentioned above, intermediate processing forms have been observed, one with a molecular mass of 81 kDa and another with a molecular mass of 74 kDa, which are thought to be generated sequentially [72,74,91]. The overall conversion of 90 kDa proMPO to the 74 kDa likely reXects removal of the aminoterminal 125 amino acid propeptide and deletion of the hexapeptide (ASFVTG) between the light and heavy subunits. Under speciWc experimental conditions, conversion of the 74 kDaintermediate to mature MPO occurs in cell-free preparations [91]. An acidic compartment is required for

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propeptide cleavage, whereas Wnal cleavage to generate mature MPO occurs optimally at a neutral pH [91]. Therefore, an acidic late endosomal compartment may be required for early but not for Wnal proteolytic trimming. The enzymes responsible for the proteolytic modiWcations of MPO precursors are unknown. As discussed previously, the M-6-P receptor system does not contribute to lysosomal targeting in myeloid cells, a conclusion derived by inferences from patients with mucolipidosis II or I-cell disease, a disorder in which the enzyme catalysing the addition of M-6-P is lacking [100], and based on studies of MPO biosynthesis [86]. Nonetheless, proMPO is normally phosphorylated to produce M-6-P residues [73,75,86] and the phosphorylated proMPO is processed rapidly into mature MPO, commensurate with the speedy traYc of granule proteins from TGN to late endosomes and secretory lysosomes. However, phosphorylated proMPO does not bind to a M-6-P receptor aYnity column [86] and alkalinization of the cell does not compromise the processing and targeting of proMPO in a way consistent with M-6-P receptor-mediated events, although these steps are somewhat slower than normal [73–77]. Moreover, alkalinization did not increase secretion of proMPO into the culture medium, a Wnding that also contrasts with the behavior of lysosomal enzymes. In the case of typical lysosomal enzymes, alkalinization inhibits M-6-P receptor targeting and increases secretion [86] because processing and targeting of these enzymes require the normal acidic milieu of the prelysosomal late endosomes [104]. Taken together, the data suggest that the targeting of MPO to the azurophilic granule is largely independent of M-6-P receptors. The contribution of the propeptide of MPO to the synthesis, structure, or activity of the mature protein is not deWned, but several possible functions have been proposed. Most investigators have pursued the hypothesis that the propeptide participates in sorting events, either directing proMPO speciWcally to the azurophilic granule for maturation or retrieving proMPO from the secretory pathway and delivering it to the granule population being synthesized contemporaneously. Given that mature neutrophils have at least three distinct granule types, each containing diVerent proteins, it is tempting to think that structural features of the granule proteins serve as speciWc targeting tags for the respective granule. However, granule sorting is by timing, not by granule-type-speciWc targeting sequences [105]. When NGAL, a speciWc granule protein, is experimentally transfected in HL-60 cells, it is directed to the azurophilic granule, i.e., the granule-class being made at the promyelocyte stage. Although these data clearly indicate that the propeptide does not seem to direct proMPO to the azurophilic granule, rather than to another granule, it does not exclude the possibility that it mediates the retrieval of proMPO from the secretory pathway. Thus the propeptide would be a “sorting sequence” in the sense that it diverts protein from the secretory pathway to the granule, but does not function to discriminate between diVerent intracellular destinations.

The function of the propeptide of MPO has been studied using two complementary strategies: examining the fate of an MPO propeptide-deleted mutant or to assess the impact of adding the propeptide to the amino terminus of a protein whose intracellular fate is known. When expressed in the 32D murine myeloid cell line, the propeptide-deleted MPO precursor is neither targeted for storage nor processed to mature MPO [103]. The fraction of the propeptide-deleted mutant MPO that is secreted has complex oligosaccharides, consistent with its traversing the TGN, however, it is rapidly degraded in the culture supernatant. The heme is inserted into the mutant, but the fraction of protein containing heme is less than that seen with normal proMPO. This is either due to ineYcient processing or instability of the resulting product of propeptide-deleted MPO [103]. When expressed in K562 cells, a human hematopoetic cell line, the propeptide-deleted mutants associate normally with CRT and CLN but fail to mature, do not incorporate heme, are inactive, and undergo rapid degradation in the proteasome [106]. Taken together, these data suggest that deletion of the propeptide compromises normal incorporation of heme and the resulting product is unstable. The instability of the product could reXect abnormal protein folding secondary to aberrant heme ligation, or the absence of a structural motif speciWc to the propeptide. Chimeric proteins tagged at the amino terminus with the propeptide were used to examine this last possibility. In Chinese hamster ovary cells, the propeptide directs some of proMPO-lysozyme hybrid to an intracellular compartment [107]. In this system, as with native MPO, the oligosaccharides on the intracellular fusion protein are high-mannose, whereas the secreted product has complex oligosaccharide side-chains. However, propeptide fusion proteins expressed in the three myeloid cell lines 32D, K562, or PLB-985, have a diVerent fate [106]. Coupling of the soluble TNF
receptor (sTNFR) to the propeptide does not redirect the fusion protein to dense intracellular organelles but rather results in retention in the ER in 32D and K562 cells. In PLB-985 cells, stably expressed GFP remains in the cytosol. Although the proMPO-GFP undergoes N-linked glycosylation in the ER at N139 in the propeptide, it fails to associate with CRT or CLN and is rapidly degraded in the proteasome. Thus, in cells that normally process endogenously expressed MPO, the propeptide alone is not suYcient to direct proMPO to its proper intracellular destination. However, its presence seems to be needed for normal processing and targeting, perhaps via intramolecular interactions not reproduced in the speciWc chimeric molecules used to date. Secreted MPO In most cells the activity of the constitutive secretory pathway is limited only by the rate of synthesis of a given protein. Although this pathway is considered a default route for proteins lacking sorting or retention signals, low

M. Hansson et al. / Archives of Biochemistry and Biophysics 445 (2006) 214–224

sorting eYciency for granule proteins in the TGN would result in increased constitutive secretion. In the case of MPO biosynthesis, secreted proMPO is monomeric, suggesting that dimer formation of mature MPO takes place only in the granule-targeting pathway or after proMPO has been compartmentalized in the granule. Alternatively, granule targeted proMPO and constitutively released proMPO may part company in the ER and travel separate routes subsequently, although no data directly support this model. The diVerent oligosaccharide processing of intracellular and secreted MPO suggests either that diversion of proMPO to the granule is proximal to the carbohydrateprocessing enzymes in the Golgi, or that access to the target oligosaccharide groups is compromised in proMPO that is destined for the azurophilic granule. The constitutively secreted precursor is 90 kDa, a fraction of which is the enzymatically active proMPO, although its physiologic function is unknown [29,76,77,102]. MPO species isolated from human plasma include both precursor and mature forms of the enzyme [29]. MPO mutations—impact on processing and targeting It is the nature of biomedical research that the study of human diseases provides lessons in normal biology and insights not likely to have been made otherwise. This paradigm applies to MPO and the lessons learned by identiWcation and elucidation of genotypes underlying inherited MPO deWciency [19,21,108]. Although Wrst believed to a be a rare disorder, inherited deWciency of MPO is relatively common in the United States and Europe, occurring with a prevalence of 1 in 2000–4000 individuals (reviewed in [109,110]), but much less common in Japan (1 in 55,000, [111,112]). Epidemiologic studies have suggested that hereditary MPO deWciency is associated with increased susceptibility to infection [113,114] and increased incidence of malignancy [115], although the clinical phenotype of increased susceptibility is not dramatic. As of January 2005, a variety of mutations resulting in MPO deWciency have been reported, including at least seven missense mutations, four of which have been characterized in detail for their impact on MPO biosynthesis [108,111,116–118]. The consequences of these genotypes on the synthesis of MPO mutant proteins are assessed using cell lines stably transfected with mutant cDNA and such studies have suggested implications for structure–function relationships of intracellular progression and targeting of MPO precursors. The Wrst genotype identiWed, and the most common in studies in Europe and the United States, is a single nucleotide mutation that results in replacement of an arginine at codon 569 in the heavy subunit with a tryptophan (R569W) [117]. When stably expressed in K562 cells, R569W is arrested at the apoproMPO stage; neither heme incorporation nor proteolytic processing and granule targeting occur. The mutant apoproMPO is degraded in the ER by a mechanism that is independent

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of the proteasome, instead the mutant apoproMPO is degraded in the ER by an unknown non-proteasomal mechanism. Whereas R569W occurs in the heavy subunit, a cysteine replaces tyrosine at codon 173 (Y173C) in the light subunit [116]. As a result of Y173C there is a novel disulWde bond created that disrupts the normally linked cysteines at codons 1 and 14 of the light subunit, as identiWed in the crystal structure [119]. Cotranslational glycosylation, association with CRT and CLN in the ER, and heme incorporation occur in Y173C. However, the proMPO(Y173C) is very unstable and is not processed to mature MPO but rather retained in the ER and subsequently degraded in the proteasome. In the Verona area in Italy, several individuals with MPO deWciency have been identiWed who have the methionine at codon 251 in light subunit replaced with a threonine (M251T) [118]. The mutant MPO precursor formed was to some extent exported out of ER [21], although neither proteolytic processing nor granule targeting was observed. Heme incorporation proceeded ineYciently and the mutant protein had corresponding low peroxidase activity. Furthermore, the mutant precursors showed prolonged association with the chaperones CRT and CLN during transit in the ER. Thus the Wndings for the M251T MPO precursors resemble those for the propeptidedeleted precursor described above [103]; low heme incorporation, constitutive secretion and lack of granule targeting. The R569W, Y173C, and M251T mutations were described in subjects from the United States and Europe. Recently the Suzuki laboratory in Tokyo has identiWed a novel genotype causing MPO deWciency, whereby a serine replaces glycine at codon 501 (G501S) [111]. The proximal histidine in the MPO heme is H502 and the adjacent glycine at position 501 is conserved among all members of the animal peroxidase family. Therefore, one would anticipate that the G501S mutation would have serious structural implications for heme formation, stability, and activity. K562 cells stably transfected with G501S synthesize both apoproMPO and proMPO, although the latter in relatively small amounts. There is constitutive secretion of 90 kDa species, but lack of proteolytic processing to mature forms [108]. The G501S precursor associates normally with CRT and CLN in the ER and has apparently normal addition and maturation of oligosaccharide side chains. Many of these features resemble those seen in the propeptide-deleted mutant [103]. The incorporation of heme but lack of enzymatic and proteolytic maturation support the proposed link between heme acquisition, aberrant folding, and faulty maturation and targeting. Folding aberrations in ER-exported proMPO, as seen in the M251T, G501S, and propeptide-deleted mutations, may obscure the interfaces that normally mediate interactions with the sorting machinery at the TGN, thus leading to secretion by default rather than proper granule targeting (Table 1).

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Table 1 Impact on processing and targeting of MPO mutations observed in individuals with inherited MPO deWciency Mutation

R569W Y173C M251T G501S

Processing of MPO mutations ER export

Heme ligation

Granule targeting

Secretion

Processed stage

Refs.

No No Low Low

No Yes Low Yes

No No No No

No No Yes Yes

ApoproMPO ProMPO ProMPO ProMPO

[117] [116] [118,21] [111,108]

The processing of these MPO mutants has been characterized after gene expression in hematopoietic cell lines.

Concluding remarks Oxygen-dependent, MPO-mediated damage to microbes contributes to optimal innate host defense, although neither the particular antimicrobial product generated in the phagosome nor the bacterial target(s) essential to achieve toxicity has been precisely deWned. Furthermore, collateral MPO damage can play a pathogenetic role in a variety of inXammatory diseases, most notably atherosclerosis. However, here too the story remains incomplete. The local origin of MPO and the source for the prerequisite H2O2 in the atherosclerotic plaque are still unresolved. The interesting hypothesis that cytokines generated in the special subintimal environment of an atheromatous plaque may initiate de novo MPO gene expression by local macrophages is provocative and merits further exploration. The complicated pathways involved in MPO biosynthesis and processing have been partially elucidated, although each novel observation seems to highlight another shortcoming in our understanding. Although the long-standing mystery of the precise nature of the heme in MPO has been solved, many unanswered questions regarding the mechanisms of autocatalytic formation of the covalent bonds to the heme prosthetic group, the evolutionary beneWts of having such covalent ligation to the heme, or the regulatory events relevant to heme acquisition in the ER remain. Explication of these events will likely provide insights broadly applicable to the other members of the animal peroxidase protein family. Mechanisms for targeting luminal granule proteins such as MPO are still unknown. Although the “targeting by timing” paradigm tightly Wts the observed experimental data, a cellular retention mechanism is required to prevent MPO loss by constitutive secretion and maintain default secretion at the low levels at which it occurs normally. Based on results from in vitro mutagenesis experiments we suggest a role for the propeptide of MPO in diverting proMPO from the secretory pathway. In this way an intact pro-piece may be necessary to achieve a conformation that is suitable for a productive interaction with the transport machinery responsible for granule targeting. The speciWc enzyme(s) that mediate proteolytic processing and the biologic advantage of dimer formation are unknown. As with all biomedical research, experiments of nature provide important clues to biologically relevant principles. Unique insights into the biosynthesis of MPO have arisen

from analyses of speciWc missense mutations that cause inherited MPO deWciency. Characterization of the functional impact of such spontaneous mutations promises to deWne further the structural determinants of MPO important in its normal biosynthesis, processing, targeting, and enzymatic activity. Acknowledgments This work was supported by the Swedish Cancer Foundation, the Swedish Childhood Cancer Foundation, and the Alfred Österlund Foundation (MH and II), and by the United States Public Health Service Grant R01 HL53592 and a Merit Review Grant from the Veterans Administration (to WMN). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

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