Characterization and Cloning of Two Isoforms of Heteroglobin, a Novel Heterodimeric Glycoprotein of the Secretoglobin-Uteroglobin Family Showing Tissue-specific and Sex Differential Expression

Javier Alvarez,Jorge Viñas,José M Martı́N Alonso,Juan Pablo Albar,Keith Ashman,Pedro Domı́Nguez

doi:10.1074/jbc.m106678200

Abstract

Heteroglobin (HGB) is a 39-kDa heterodimeric protein detected under non-reducing conditions in harderian, parotid, and submaxillary glands and saliva of the Syrian hamster with antiserum raised against the carboxyl end deduced from the female harderian gland cDNA FHG22 (Dominguez, P. (1995) FEBS Lett. 376, 257-261). After reduction, only one 5.6-kDa polypeptide, named HGB.A, was immunodetected and identified by sequencing as the mature FHG22 product. Tissue-specific expression of HGB.A and HGB mimics that of FHG22 mRNA, with sex differences in submaxillary and harderian glands. Purification of HGB revealed it consists of HGB.A disulfide bonded to HGB.B, a 33.5-kDa N-glycosylated subunit that yields a 9-kDa core polypeptide after deglycosylation. Two highly homologous (96.2%) cDNA clones (HGB.B1 and HGB.B2) encoding 94 amino acid-long isoforms were identified by screening a female harderian gland library with an HGB.B probe. The corresponding mature polypeptides are 78 amino acids long with 12 differences, but 3 putative N-glycosylation sites are maintained. The expression of HGB.B mRNAs is parallel to that of HGB and HGB.A, but no HGB.B2 mRNA was detected in submaxillary glands. Homology studies indicate that HGB.A and HGB.B1/HGB.B2 belong to different subfamilies of the secretoglobin-uteroglobin family and form heterodimers as previously described.

Highlights

The existence of a uteroglobin/Clara cell 10-kDa family of proteins including UGB/CC10 orthologs and paralogs (such as subunits of rat prostatein, cat Fel d 1 and mouse androgenbinding protein, and cDNAs like hamster FHG22) was previously suggested [1,2,3,4]
The existence of a uteroglobin/Clara cell 10-kDa family of proteins including UGB/CC10 orthologs and paralogs was previously suggested [1,2,3,4]
Heteroglobin (HGB) is a 39-kDa heterodimeric protein detected under non-reducing conditions in harderian, parotid, and submaxillary glands and saliva of the Syrian hamster with antiserum raised against the carboxyl end deduced from the female harderian gland cDNA FHG22 (Domınguez, P. (1995) FEBS Lett. 376, 257–261)

Summary

Introduction

The existence of a uteroglobin/Clara cell 10-kDa family of proteins including UGB/CC10 orthologs and paralogs (such as subunits of rat prostatein, cat Fel d 1 and mouse androgenbinding protein, and cDNAs like hamster FHG22) was previously suggested [1,2,3,4]. Enzymic Deglycosylations—In the study of unpurified HGB deglycosylation, female hamster parotid, submaxillary, and harderian gland homogenates or saliva were digested with endoglycosidases to remove saccharides from the polypeptide chains, and the protein was immunodetected with AF22P3.

Results

Conclusion