Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.

The AMP deaminase-associated variant of histidineproline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zincbinding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn 2؉ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-prolinerich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zincbinding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase. Histidine-proline-rich glycoprotein (HPRG)1 is an approximately 70-kDa glycoprotein that is present at a relatively high concentration in the plasma of vertebrates. Although the physiological role of this protein remains unclear, it has been implicated in a number of processes, including blood coagulation and fibrinolysis, immune response, and transport of metal ions (1). The cellular origin of the mouse protein has recently been defined by Northern blot analysis showing that the HPRG mRNA is localized specifically to the liver, suggesting that the previously described HPRG expression by immune cells is due to the acquisition of the plasma protein derived from the liver (2). In a previous paper we reported that denaturation of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) in acidic medium allows the chromatographic separation from the enzyme of a peptide with an amino acid composition significantly different from that derived from the available AMP deaminase cDNAs. N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of the novel protein to rabbit plasma HPRG although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension (3). We now report that the AMP deaminase-associated variant of HPRG can be isolated from rabbit skeletal muscle by a modification of the protocol used in our laboratory for the purification of AMP deaminase. The modification allows the partial dissociation of HPRG at a high degree of purity from the cellulose phosphate-bound enzyme.Rabbit plasma HPRG contains 53 histidine residues, of which 34 are located in the histidine-proline-rich d...

Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

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SUMMARY Histidine-proline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP deaminase (AMPD). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers. Histidine-proline-rich glycoprotein (HPRG) is a protein present at a relatively high concentration in the plasma of vertebrates. Its specific function remains unclear, although it has been implicated in several phenomena, including blood coagulation and fibrinolysis (Peterson et al. 1987). In a recent article we reported the isolation from purified rabbit skeletal muscle AMP deaminase (AMPD) of an approximately 75 kD novel peptide with an amino acid composition significantly different from that derived from the available AMPD cDNAs (Ranieri-Raggi et al. 1997). N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of this protein to rabbit plasma HPRG (Borza et al. 1996) although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension. Among the 60 amino acids sequenced up to now in the novel HPRG isoform, four substitutions were found with respect to the published rabbit HPRG sequence, all of them localized in the 472-477 region that also differs in five amino acid residues compared with the homologous region of the human protein (residues 461-466) (Koide et al. 1986). This divergence enabled us to raise a rabbit antibody against a synthetic peptide equivalent to residues 462-471 of human plasma HPRG. The similarity between the 461-466 region of human plasma HPRG (S-F-P-L-P-H) and the corresponding sequence of the HPRG-like molecule isolated from rabbit skeletal muscle (S-F-S-L-R-H) prompted us to utilize the antibody to probe the immunohistochemical localization of the putative HPRG-like protein in human skeletal muscle. The experimental results show that the anti-HPRG antibody reacts with an AMPD preparation from human skeletal muscle. Moreover, a clear positive reaction was detected at the level of Type IIB fibers, giving evidence of the presence of an HPRGlike peptide in human skeletal muscle. These observations suggest a correlation of this protein as well as Correspondence to: Prof.