Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation.

F Lanza,M Morales,C De La Salle,J.P Cazenave,K.J Clemetson,T Shimomura,D.R Phillips

doi:10.1016/s0021-9258(19)36855-3

F Lanza, M Morales + Show 5 more

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https://doi.org/10.1016/s0021-9258(19)36855-3

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Abstract

Glycoprotein V (GPV) is a major platelet membrane 82-kDa glycoprotein, missing in the Bernard-Soulier syndrome, that is cleaved when platelets are treated with thrombin. We report the cloning and sequencing of the GPV cDNA and gene obtained by a combination of polymerase chain reaction amplification of platelet mRNA and genomic library screening. The single-copy gene for GPV is contained within 6.5 kilobase pairs (kb) of genomic sequence and has a simple structure with a single intron of 958 base pairs in the 5'-untranslated sequence; the coding sequence is contained within a single exon. The promoter region contains a canonical TATA box, and putative GATA, Ets-1, and Sp1 cis-acting elements. Reverse transcription-polymerase chain reaction analysis on RNAs from cells of different hematopoietic origins revealed that GPV was specifically transcribed from platelets and from cells of the megakaryocytic lineage (megakaryocytes, HEL cells). A single transcript of 4.5 kb for GPV was detected in human platelets by Northern blot analysis. The entire amino acid sequence of GPV was deduced from the cDNA and genomic sequences. Mature GPV was composed of 544 amino acids which contained a single transmembrane domain, a short cytoplasmic domain (16 residues), and a large extracellular domain with 8 potential N-glycosylation sites. Analysis of the extracellular domain revealed the presence of 15 tandem Leu-rich repeats of 24 amino acids with homology to GPIb alpha and identified a cleavage site for thrombin near the COOH terminus with similarity to the A alpha chain of fibrinogen, but no hirudin-like sequence was found.

Highlights

We report thecloning and sequencingof the GPVcDNA and gene obtained by a combination of polymerase chain reaction amplification of platelet mRNA and genomic library screening
Analysis of the GPV extracellular sequence revealed the presence of 15 leucine-rich tandem repeats of24 amino acids (Fig. 6).These repeats are very similar to repeats found in platelet GPIba, GPIbp, and GPM and to the 24-amino acid consensus sequence based on the repeats found in othermembers of the leucine-rich glycoproteins (LRG) family.The last GPV Leu-rich repeat is flanked
This study describes the cloning and sequencingof the cDNA and gene of platelet GPV

Summary

RESULTS

748-bp 32P-labeled GPV cDNA fragment (see Fig. la for the location of the probe) a t 45 "C overnight. Exon 2 contained 2 bpof 5'-untranslated sequence, 1,168 bp of coding sequence obtained by PCR, and an additional512 bp in frame coding sequence before reaching a TAA stop codon Translation of this additional sequencerevealed the presence of a peptidesequence (thl) corresponding to the newly exposed NHZ-terminal sequence of thrombin-cleaved GPV. Analysis of the GPV extracellular sequence revealed the presence of 15 leucine-rich tandem repeats of amino acids (Fig. 6).These repeats are very similar to repeats found in platelet GPIba, GPIbp, and GPM and to the 24-amino acid consensus sequence based on the repeats found in othermembers of the LRG family.The last GPV Leu-rich repeat is flanked. The new NH2-terminal sequence revealed by the potential cleavage site matched that of the Thl peptide obtained after NH2-terminal sequencing of thrombincleaved platelet GPV [20, 21]

DISCUSSION

C CACGTGTGGG CA

Findings

LR repeats